Enzymes as drugs have two important features that distinguish them from all other types of drugs. First, enzymes often bind and act on their targets with great affinity and specificity. Second, enzymes are catalytic and convert multiple target molecules to the desired products. These two features make enzymes specific and potent drugs that can accomplish therapeutic biochemistry in the body that small molecules cannot. These characteristics have resulted in the development of many enzyme drugs for a wide range of disorders.
Yeasts are single-celled microorganisms that are classified, along with molds and mushrooms, as members of the Kingdom Fungi (Alfenore et al., 2002 and Alfenore et al., 2004). Although yeasts are unicellular organisms, they possess a cellular organization similar to that of higher organisms, including humans. Specifically, their genetic content is contained within a nucleus. This classifies them as eukaryotic organisms, unlike their single-celled counterparts, bacteria, which do not have a nucleus and are considered prokaryotes (Barnett et al., 2000 and Boekhout et al., 2002).
Yeast is widely dispersed in nature with a wide variety of habitats. They are commonly found on plant leaves, flowers, and fruits, as well as in soil. Yeasts are also found on the surface of the skin and in the intestinal tracts of warm-blooded animals, where they may live symbiotically or as parasites. Yeasts are responsible for several types of infections including oral thrush, vaginitis, urinary tract infection, endocarditis, respiratory syndromes, meningitis, etc. The common “yeast infection” is typically caused by Candida albicans. Beside infections, yeast is very useful in commercial application. Yeast has long been considered to be the organism of choice for the production of alcoholic beverages, bread, and a large variety of industrial products (Alfenore et al., 2002 and Abouzied and Reddy, 1986). This is based on the ease with which the metabolism of using genetic techniques, the speed with which it can be grown to high cell yields (biomass), the ease with which this biomass can be separated from products and the knowledge that it is generally recognized as safe (GRAS).
Yeasts are unicellular fungi that have mostly used in the fermentation process for the production of food and alcoholic beverages. Yeasts are identified through the physiological characters. Yeasts are mostly found in natural habitats viz., plant leaves, flowers, soil and salt water or some skin surfaces and intestinal tracts of warm blooded animals, where they may live symbiotically or as parasites.
Yeasts are a polyphyletic group of basidiomycetous and ascomycetous fungi with a unique characteristic of unicellular growth. The term ‘yeast’ is derived from the Old Dutch word gist and the German word gischt, which refers to fermentation. There are approximately 100 genera and 800 described species of yeasts and estimates suggest that these numbers represent only about 1% of the species that exist in nature, the rest being non-culturable.
Yeasts have been used by the food industry principally for the production of ethanol and carbon dioxide, which are important to the brewing, wine distilling and baking industries. Their environmental role is similar to many other fungi, acting as saprophytes by converting plant and animal organics to yeast biomass and by-products, which may have commercial importance. Some yeast are pathogenic to plants and animals. Yeasts are rich with proteins, lipids and vitamins. Biotransformation of raw material into yeast biomass (single-cell protein) is highly significant, due to the nutritional quality of yeast and its possible utilization as animal or aquaculture feed. Yeasts also have immunostimulatory properties by virtue of their complex carbohydrate and nucleic acid components. They can be produced very efficiently and economically because of their shorter generation time and use of inexpensive culture media. Lipids, pullulans and enzymes from yeasts are extracellular metabolites of commercial importance.
Today, there are about 500 species of yeasts in 60 genera and about 1,000 species of yeast-like organisms in the world. Although the fungi are multicellular, growing as filaments called hyphae, the yeasts or the yeast-like cells have morphological terms that refer to single celled fungi. The yeasts have already been used for various industrial purposes and basic studies on molecular biology, genetics in addition to traditional baking and alcoholic fermentations.
Among the yeast species, Saccharomyces cerevisiae has been thought to be one of the most important micro-organisms for humans because most of the yeasts applying to various fermentation technologies were identified as the S.cerevisiae which had been secured as safe for foods by experience for a long period. The S. cerevisiae strains have the highest fermentative activities among the yeast species and they are found in various natural environments such as flowers, trees, animals, soils, hydrosphere and artificial environments such as foods, drinks (Barnet et al., 2000).
Since the beginning of the 21st century, various studies on marine-derived yeasts except for S. cerevisiae have been reported (Sreedevi et al., 2008; Chen et al., 2009; Mastuda et al., 2008; Zhang et al., 2010). Otherwise, only a few studies have been reported about isolation and application of marine-derived S. cerevisiae as a main target (Saravanakumar et al., 2013). A marine-derived S. cerevisiae C19 with the highest fermentative activity among many yeast isolates was isolated and applied to the ethanol production from various biomasses (Takagi et al., 2015; Obara et al., 2015; Obara et al., 2012).
Enzymes play a definitive role in the production of wine, which could be seen as the product of enzymatic transformation of the grape juice. The enzyme activities do not only originate from the grape itself, but also from yeast and other microorganisms. The winemaker now reinforces and extends the action of these endogenous enzymes by the use of exogenous, industrial enzyme preparations (Fleet, 1993). The production of extracellular hydrolytic enzymes by indigenous yeast could be significant and needs to be understood and managed to the benefit of wine production. Moreover, wine yeast could be potential sources for the commercial production of enzymes to be used in the process of winemaking (Charoenchai et al., 1997).
Enzymes are biological catalysts which are an indispensible component of biological reactions. The use of chemical catalysts has been followed for a very long time. Chemical catalysis though widely used was very cumbersome. The disadvantages that this method poses include need for high temperature and pressure for catalysis and the moderate specificity. These limitations were overcome by the use of enzymes. Enzymes work at milder conditions when compared to that required by chemical catalysts for operation. Also enzymes are highly specific and catalyze reactions faster than chemical catalysts (Prasad Nooralabettu Krishna, 2011).
Enzymes are now being used in various sectors of industry. They are used in detergents; paper industry, textile industry, food industry and many others industrial applications. Enzymes have been in use since ancient times and they have been used in saccharification of starch, production of beverages like beer, treatment of digestive disorders and production of cheese from milk (Drauz et al., 2012). Among the many enzymes that are widely used ?-Amylase has been in increasing demand due to its crucial role of starch hydrolysis and the applications of this hydrolytic action. The following sections elaborate on the types of amylases and their roles in enzymatic reactions.
Saccharomyces cerevisiae, the principal wine yeast, is not recognized as a significant producer of extracellular enzymes, although a few strains have recently been reported to degrade polygalacturonate (McKay, 1990). There is little information on the production of extracellular enzymes by non-Saccharomyces wine yeast, although some strains of Kloeckera apiculata show extracellular protease activity (Lagace and Bisson, 1990; Dizy and Bisson, 2000). Various authors have reported glycosidase production by S. cerevisiae and the potential for these enzymes to enhance wine favor (Delcroix et al. 1994).
The following objectives are:
Analysis of physicochemical parameters of water samples
Analysis of physicochemical properties of soil samples
Isolation of yeast from marine water samples of Muthupet area
Isolation of yeast from marine soil samples of Muthupet area
Identification of yeast by using morphology and biochemical test
Screening of extracellular enzymatic activity from the yeast isolates
Quantitative analysis of extracellular enzymes from the potential isolates of yeast
Qualitative and quantitative analysis of bioactive compounds from potential yeast
Optimization of enzymatic activity of different potential strains of yeast
Amplification of yeast by using PCR
Gene sequencing and deposition
2. REVIEW OF LITERATURE
Seawater is cheap and contains a spectrum of nutritional minerals – can be a promising alternative to freshwater with the possibility of improving the overall economics of the process and contributing to overcoming freshwater crisis. Also, the high amount of salts in seawater can serve as selective agent against the microbial contamination in the biorefineries, adding one more favour to the proses. Therefore, employing marine derived yeasts in industry seems to be important as they are able to propagate and produce their metabolites on seawater based medium. Over the last century, terrestrial yeasts have received great attention due to their ability of producing many types of bioactive compounds. On the other hand, the handful research on marine yeasts that conducted in recent years proved that the marine yeasts have several promising features over the terrestrial ones, e.g. higher tolerance and higher productivity. This review gathers the most recent techniques used for marine yeast isolation and focuses on the latest achievement of marine yeast in industry. Also, it indicates the high potential of marine yeasts for the industry sector and their superiorities over the terrestrial ones (Zaky and Du, 2013).
Morphological identification of yeast
The 240 yeasts isolated from soils of the Maraca Ecological Station in the Brazilian Amazon were identified and screened for mycocin production. These strains included 82% of ascomycetous and 18% basidiomicetous affinities and the prevalent species were Candida etchellsii, Candida famata, Candida robusta, Candida rugosa, Candida valida, Debaryomyces hansenii, Cryptococcus albidus, Cryptococcus laurentii, Rhodotorula glutinis, Rhodotorula minuta and Rhodotorula mucilaginosa. Mycocins able to kill some yeasts were produced by 6 strains identified as Issatchenkia sp., Saccharomyces exiguous, Williopsis saturnus, var. subsufficiens and W. saturnus (Marcos et al., 2002).
Muhammad Mushtaq et al. (2004) studied that the identification of morphological and physiological/biochemical characteristics, 4 genera and 5 species of yeasts were isolated and identified from cultivated soil and 16 species belonging to 12 genera from garden soil. The identified yeast species included anamorphic and teleomorphic Ascomycetes and Basidiomycetes which have not been reported from Pakistan.
Yeast is a group of fungi in which unicellular form is predominant. Most of the yeasts are represented in Sub Division Ascomycotina and Basidiomycotina of the kingdom Mycota. As a group of microorganisms yeasts have cosmopolitan distribution. They have been isolated from natural substrates like leaves, flowers, sweet fruits, grains and fleshy fungi, exudates of trees, insect, dung and soil. They play their role in the dynamics of biological and chemical turnover in soil, plants, animals and water. There are about 100 genera and 700 species of yeasts of which only 5 genera and 7 species have been reported from Pakistan. The yeast mycoflora present in two different kinds of soil. These have been identified up to species level on the basis of their morphological and physiological/biochemical characteristics (Muhammad Mushtaq et al., 2004).
Wang et al. (2007) reported that the isolated a total of 427 strains from different marine substrates and their lipase activity was estimated. They found that nine yeast strains obtained in this study when grown in a medium with olive oil could produce lipase. The optimal pH and temperature of the lipases produced by them were between 6.0-8.5 and 35-40°C respectively. Some lipases from the yeast strains could actively hydrolyse different oils, indicating that they may have potential applications in industry.
The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island) and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano) soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia), Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula and Sporidiobolus and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species (Vaz et al., 2011).
The isolation and identification of yeast from Manihot esculenta, Zea mays, Cola acuminata and Sorghum bicolor was done using the spread plate technique. Morphological, cultural, physiological and molecular characterizations were carried out resulting in etermination of the species. Four isolates belonging to different genera which include Pichia, Kluyveromyces, Candida and Saccharomyces were identified. This present study showed that the yeast isolates have the potential to ferment both hexose and pentose sugars (Abosede Margaret Ebabhi et al., 2013).
A total of thirty yeast strains were isolated from various soil sources in PYG medium. The isolates were screened for the activity of nitrogen fixation on nitrogen fixing yeast medium. Among all yeast isolates, N3, N18, N21 and N24 could grow well in this medium. According to their morphological characteristics, sugar assimilation and fermentation patterns and some biochemical characteristics, the selected yeast isolates could not be identified exactly. N3, N18 and N24 accumulated highest amount of ammonium concentration in raffinsoe containing media by giving 9.365 ppm, 4.774 ppm and 5.222 ppm of ammonium concentration. But, the highest amount of ammonium concentration accumulated by N21 was 2.263 ppm in the mannitol containing medium. In nitrogen free mineral broth medium, accumulated ammonium concentrations of selected yeast isolates were increased. N3, N18 and N24 accumulated 11.866ppm, 5.521 ppm and 8.027 ppm of ammonium concentration respectively in raffinose and N21 accumulated 5.089ppm of ammonium concentration in Mannitol. On detection of accumulated ammonium concentration by Viscolor Alpha Ammonium Detection Kit, it was also found that they gave color development. In a pot trial study, maximum total nitrogen content in sorghum plant was recorded from inoculated treatment of N3 (1.52%) followed by inoculated treatment of N24 (1.51%) (Nwe Ni Win Htet et al., 2013).
Obasi et al. (2014) studied that the 98 strains of yeast were isolated from fresh or healthy fermented and defective orange fruit. A total of 51 species were identified using AP120C, representing 4 genera; 32 from fresh fermented orange juice (FSSOJ) which 19 species were from defective orange juice (DSSOJ). Among (FSSOJ) isolates, Candida kruesi and Rhodotorula minuta were the predominant species, while Candida zeylanoides and Candida parapsilosis were the dominant species in DSSOJ. Candida and Rhodotorula species were both isolated from fresh or healthy and defective orange juice, while Kodamaea and Geotrichum species were isolated from the fresh or healthy orange juice only. Candida Kruesi had the highest prevalence (57%), Rhodotorula minuta (20%), Candida zeylanoides (8%), Candida parapsilosis (6%), Geotrichum capitatum (4%), Candida norvegensis and Kodamea (1%) respectively. Candida lusitaniae, Candida parapsilosis and Rhodotorula minuta metabolised xylose which showed that they possess genes responsible for xylose fermentation. The diversity of yeast isolated from the FSSOJ and DSSOJ showed that orange juice employ a whole range of natural flora that could function under varied environmental conditions
The yeasts are eukaryotic microorganisms which can also be used for bioethanol production. In this modern era of increasing demand for energy and fuel, the microbial biosynthesis of ethanol has gained importance. The potential yeasts for ethanol production from pentose and hexose sugars were identified. Yeasts were isolated from soil and different food samples. They were identified and characterized based on cell morphology (e.g., mode of cell division and spore shape) and physiology (e.g., sugar fermentation tests). Six different species of yeasts were cultured in three sets of broth for 24, 48, 72, and 96 hours for bioethanol production. The yeasts isolated from black and green grapes relatively synthesized higher concentration of ethanol (Sandeep Thapa et al., 2015).
Four sea water samples were collected from different locality of Saviyarmunnai, Vavalthottam, Manakkattai and Devararul Muthupet mangroves environs by using YM medium was used. A total of 13 isolates were isolated such as Aureobasidium nulluns, Candida elaebora, Candida sake, Candida zeylanoides, Cryptococcus hansenii, Candida victoriae, Dioszella crocera, Dioszella aurantiace, Leucosporidilla fragaria, Leucosporidilla auscorum, Rhodotorula glacialis, Rhodotorula larvngis and Saccharomyces cerevisiae was frequently distributed from Saviyarmunnai, Vavalthottam, Manakkattai and Devarraul villages of Muthupet environs were identified respectively (Jayalakshmi and Umamaheshwari, 2016).
The isolation, identification and assessing the dough fermenting abilities of yeasts from various indigenous sources which can potentially be employed as a leavening agent. Differential test were applied including cultural, morphological and biochemical characteristics (using API20C AUX Kit (BioMeriux), which facilitated the identification of the yeasts to specie level. The isolates were subjected to baking potency test; ethanol tolerance test, hydrogen sulphide (H2S) production test, temperature tolerance test, flocculation test (effect of shaking) and leavening action on bread dough. Thirteen (13) yeasts were isolated from sweet orange (Citrus sinensis), pineapple (Ananas comosus) and palm wine. These isolates belong to the genera of Candida, Rhodotorula, Kodamaea and Cryptococcus. Four yeast Species namely; Candida colliculosa, Candida krusei, Rhodotorula mucilagnosa and Rhodotorula minuta were used to ferment wheat flour dough in order to determine their individual fermentative abilities where Rhodotorula minuta and Rhodotorula mucilagnosa showed better performance compared to commercial yeast. Thus indicates that the local fruit could be a potential source of indigenous yeast species for leavening agent in bread making (Maryam et al., 2017).
Physicochemical properties vs yeast isolates
Principle component analysis and multiple linear regression using stepwise selection was used to model the relation between abiotic variables (principle component 1 and principle component 2 scores) and yeast biodiversity (the number of species present at a given site). These analyses identified soil pH and electrical conductivity as significant predictors of yeast biodiversity (Connell et al., 2007).
The physico-chemical properties and their inter-relationship were studied in twenty seven profile representing three profiles from each topo-sequence (up, medium and low land) of three agro-climatic zones of Jharkhand viz., (i) central and north-eastern plateau i.e. zone-IV, (ii) western plateau i.e. zone-V and (iii) south eastern plateau i.e. zone-VI. The soil samples were analysed for various physico-chemical properties viz., organic carbon, pH, EC, CaCO3, CEC, clay and silt content, using standard laboratory procedures. Analysis of soil pH, organic carbon and calcium carbonate revealed that soil pH and CaCO3 increased with increasing soil depth of profiles. On contrary, organic carbon of the soils declined with increasing depth. Higher values of CEC in sub-surface horizons commensurate with the amount of clay. Variation of soil pH and EC was less in lowland and upland profiles, respectively whereas in case of CaCO3, upland profiles show maximum variations. Correlation matrix indicated that soil pH were significantly correlated with CaCO3 (r=0.72**) and organic carbon (r= – 0.38**). Clay were positively and significantly correlated with CEC (r= 0.64**) and EC (r= 0.50**). Calcium Carbonate were significantly correlated EC(r= 0.35**) and organic carbon (r= -0.44) (Rakesh Kumar et al., 2012).
Patil et al. (2012) reported that the physico-chemical parameters such as color, temperature, acidity, hardness, pH, sulphate, chloride, DO, BOD, COD, alkalinity used for testing of water quality. Some water analysis reports with physic-chemical parameters have been given for the exploring parameter study. Guidelines of different physic-chemical parameters also have been given for comparing the value of real water sample.
Das and Bindi, (2014) conducted that the physicochemical analysis of soil of Jaisamand lake area. The physical and chemical parameters like pH, EC, moisture content, organic matter, nitrogen, potassium and phosphorous were studied, tabulated and briefly discussed. The soil of Jaisamand is of black cotton type with reddish brown colour. The moisture content of Jaisamand soil is high i.e. 72.41% as compared to normal agricultural field (32.27%). The colour, alkalinity, insufficient amount of phosphorous and organic carbon indicate the starting of soil contamination from agricultural and domestic sewages. The data obtained clearly indicated that the lake is oligotrophic in nature and that the area is good for agriculture of wheat, gram, mustard, zea maize, cotton etc.
The impact of agrochemicals on soil physicochemical, soil microbial population and hydrolytic enzyme activities were evaluated at three different soil depths in the Imo River Basin farm (IRBF) and compared with soil from a farmland located about 200 meters away from it, which served as the control. Soil physicochemical parameters, hydrolytic enzyme activities and microbial bioload were determined using standard methods. The significant reduction (P < 0.05) in electrical conductivity, moisture content and cation exchange capacity. Organic matter, pH, phosphate, sulphate, nitrates and potassium in the test samples increased significantly (P<0.05) compared with the control. There was a significant reduction in enzyme activities (P < 0.05) of the dehydrogenase, polyphenol oxidase, hydrogen peroxidase, acid phosphatase and alkaline phosphatase, in the test samples compared to control. There was also a significant increase (P < 0.05) in urease activity in the test soil especially at the topsoil compared to control. Soil microbial types and population were significantly reduced (P < 0.05) in the test soil compared to control. The result shows that agricultural activities affects soil properties adversely and calls for control of some agricultural practices (Leo et al., 2014).
The quality of water from four coastal towns (Ayetoro, Idiogba, Bijimi and Asumogha) in Ilaje local government area of Ondo State was assessed using standard methods with the view of determining the level of pollution through anthropogenic activities and state of the aquatic ecosystem. The results of the analyses of the water samples showed that Dissolved Oxygen (DO) had the highest mean of 7.66 mg/l in Ayetoro while the lowest mean (7.53 mg/l) was recorded in Bijimi; Temperature had minimum mean value of 29.42 oC in both Bijimi and Idiogba and maximum mean of 29.75°C recorded in Asumogha. The minimum mean of pH across the four locations was recorded in Asumogha (6.63) and the maximum mean was recorded in Idiogba (6.71). The conductivity of Idiogba had the least mean value of 41.00 ?S/cm and Ayetoro had the highest mean value of 41.83 ?S/cm. Salinity ranged from 16.35 o/oo in Asumogha to 16.65 o/oo in Idiogba and the minimum mean of hardness (84.57 mg/l) was recorded in Asumogha while the maximum mean of 87.16 mg/l was recorded in Ayetoro. Also, turbidity ranged between 41.95 NTU in Bijimi and 45.36 NTU in Asumogha. The physico-chemical parameters of water determined (except turbidity and hardness) showed no significant difference across the four sampling stations at P<0.05 level of significance (Olatayo, 2014).
The physicochemical properties of soil (0-0.20 m) were analysed, the characteristics of tea growing soils under three different locations of West Bengal (India), considering the age and elevation of tea plantation. The soil samples of Dooars and Terai region of West Bengal were collected on the basis of age (young, medium and old) of the tea plants, while that from Darjeeling region as organic and non-organic tea growing soils. The organic and non-organic tea soils were collected on the basis of elevations of the sites. The soils of the Dooars region were clay to sandy loam in texture whereas, soils of Terai and Darjeeling were sandy loam in texture. The selected soils were strong to moderately acidic in reaction with low electrical conductivity (EC), Ca+2 and Mg+2 content. The organic carbon content of different regions was found medium to high, but very little variation was obtained with organic tea growing regions of Darjeeling. The soil available N and P content were low to medium in all the regions but higher available K content were found with the soils of Dooars and Terai regions. The cation exchange capacity (CEC) of soils varied from low to medium. The correlation study indicated that CEC, available N and K were influenced by soil organic carbon content, while the available P, Ca+2 and Mg+2 content by the soil pH. The available N, K, EC and CEC were negatively influenced by sand content of the soils (Ray and mukhopadhyay, 2015).
Sreenivasulu et al. (2015) evaluated that the physico-chemical characteristics like pH, temperature, EC and chemical parameters such as Dissolved Oxygen, Organic matter and Silica provides valuable information on the quality of the water, the source(s) of the variations and their impacts on the functions and biodiversity of the water body. The study area (Tupilipalem coast) is located on the northern most side of the Pulicat Lake in Nellore district, Andhra Pradesh, which is a proposal site for construction of major port (Dugarajapatnam Port). A systematic study has been carried out to assess the water quality status of the study area. A total of 12 water samples were collected during October, 2014 and analyzed for physico- chemical parameters (pH, bottom water temperature, air temperature, EC, Salinity, Dissolved Oxygen, Organic matter, and Silica). Statistical analysis like Pearson Correlation matrix and Factor loadings were performed to the data set to know the relationship among the studied parameters.
Physicochemical properties of water were determined according to the standards of the American Public Health Association. Generally, all those parameters were recorded a small variation between stations. The variation in physico-chemical parameters like salinity, temperature, dissolved oxygen and pH at Gwadar (Coastal water of Balochistan) were recorded. The range of air temperature of coastal water of Balochistan during 2004 and 2006 varies from 25 ºC to 37 ºC, water temperature ranged from 15.00 ºC to 33.00 ºC, pH ranged from 7.08 to 8.95, salinity ranged from 37.4‰ to 41.3‰ and dissolved oxygen ranged from 5.32 to 8.67 mg/L. The parameters of Balochistan coast of Pakistan is not dangerous for marine habitat and the use of these parameters in monitoring programs to assess ecosystem health has the potential to inform the general public and decision-makers about the state of the coastal ecosystems. To save this vital important habitat, the government agencies and scientists should work with proper attention (Elahi et al., 2015).
The coastal zone of Visakhapatnam is receiving a sewage waste and industrial effluents owing to intensified industrial and population growth. The physicochemical parameters rainfall, atmospheric temperature, water temperature, pH, salinity and dissolved oxygen seasonal variations were discussed. pH ranges between 7.5 to 8.3. It was minimum in July and was highest in October. However, DO was maximum in May (6.7mg/L) while it was minimum in the month of September (4.0 mg/L) (Ramana et al., 2015).
Mangrove soil condition was essential factor for mangrove reforestation and coastal rehabilitation. The marine environment is a prolific resource for the isolation of microorganisms. The analysis of physicochemical characteristics and bacterial populations in sediment soil at Karankadu mangrove forest, Ramanathapuram Dist in Tamil Nadu for a period of one year during April 2013 to March 2014. Physical parameters like pH, Electrical conductivity (EC), Organic carbon (%), Temperature, Salinity, Soil texture, Color, and Chemical parameters of Phosphorus, Potassium, Calcium, Iron, Nitrogen, Manganese, Zinc were analyzed in four seasonal intervals. The nature of soil texture is characterized by the abundance of clay loam. The serial dilution plate technique was employed to enumerate the sediment soil bacteria. Correlation coefficient between the physicochemical parameters of soil and total number of isolated bacterial colonies (Saseeswari et al., 2015).
The physical and chemical characteristics of soil in three different marine ecosystem of six different places sea shore (Point Calimere Kodiyakarai, Adirampattinam, Mallipattinam and Manora), saltpan (Vedaranyam), mangroves Forest (Muthupet) Palk Strait coastal regions of Tamilnadu, India, was performed. The marine soil were selected for the following boundaries like Soil texture, Calcium Carbonate, Electrical conductivity, Power of hydrogen, Macronutrients like (Organic carbon, Nitrogen, Phosphorus, Potassium), Micronutrients like (Iron, Manganese, Zinc, Copper) and others Caution exchange capacity, Magnesium, Sodium were studied. At the end of the soil collected from various stations showed differences in all analyzed features (Manikandan and Vijayakumar, 2016).
The marine ecosystem of the city of Sidi Ifni, not yet studied and some sites still receive untreated wastewater. Six samplings campaigns were conducted during January-June 2015 in Mirleft, Cheikh and Sidi Ifni waste water treatment plant (Step) sites. The physicochemical parameters measured in coastal waters (T°C, pH, Dissolved O2, Salinity, Conductivity, Turbidity, Salinity, Chlorides, Sulfates, Phosphorus, Ammonia Nitrogen, Nitrate and Nitrite), and especially in sites that receive untreated wastewater from Sidi Ifni coasts (Cheikh and Step sites) compared to the reference site Mirleft. The impact of this disruption and requires the strengthening of the surveillance of the coast of Sidi Ifni in the framework of a monitoring strategy and effective prevention against marine pollution (Abbassi et al., 2017).
Paskevicus (2001) studied that the yeast strains produce lipase enzyme. The most active lipase producers belonged to the genera Rhodotorula, Candida, Pichia and Geotrichum. Lipases catalyse a wide range of reactions like hydrolysis, esterification, alcoholysis, acidolysis, aminolysis etc. Lipases are mainly involved in detergent industry and biodegradation, especially oil residues. Yeast enzymes were found to be useful in various industrial processes which emphasize their direct contribution to our day to day life. These enzymes are produced mostly extracellular by different metabolic reactions taking place inside the cell and participate in various transformation activities like mineralization of organic compounds (Hasan et al., 2006).
Strauss et al. (2001) studied that the 245 yeast isolates, belonging to the genera Kloeckera, Candida, Debaryomyces, Rhodotorula, Pichia, Zygosaccharomyces, Hanseniaspora and Kluyveromyces were screened for the production of extracellular pectinases, proteases b-glucanases, lichenases, b-glucosidases, cellulases, xylanases, amylases and sulphite reductase activity. These yeasts, representing 21 species, were previously isolated from grapes and clari®ed grape juice. The production of all extracellular hydrolytic enzymes screened for was observed except b-glucosidase activity. The amount and range of enzymes produced varied with different isolates of the same species.
In 348 yeast (193 ascomycetes and 155 basidiomycetes) and 46 yeast like strains (Aureobasidium pullulans) were screened for their EEA profile. The spread occurrence of extracellular amylases, esterases, lipases, proteases, pectinases and chitinases appeared to be a strain-related character. Yeasts isolated from tropical environments could represent a promising source of extra cellular enzymatic activity. Selected strains showed maximum levels of EEA under acidic or neutral conditions. Significance and Impact of the Study: This study demonstrated the potential for yeasts isolated from extreme environments as sources of industrially relevant enzymes for biotechnological purposes (Buzzini and Martini, 2002).
Yeast amylases have many applications in bread and baking industry, starch liquefaction and saccharification, paper industry, detergent industry, medical and clinical analysis, food and pharmaceutical industries (Chi et al., 2003; Gupta et al., 2003). Amylolytic yeasts convert starchy biomass to single cell protein and ethanol (Li et al., 2006). A protease producing strain isolated from the sediments of saltern near Qingdao, China, had the highest activity at pH 9 and 45ºC (Chi et al., 2007). This principal enzyme, protease has many applications in detergent, leather processing and feed industry besides waste treatment (Ni et al., 2008).
Wang et al. (2007) isolated a total of 427 strains from different marine substrates, and their lipase activity was estimated. They found that nine yeast strains obtained in this study when grown in a medium with olive oil could produce lipase. The optimal pH and temperature of the lipases produced by them were between 6.0-8.5 and 35-40ºC respectively. Some lipases from the yeast strains could actively hydrolyse different oils, indicating that they may have potential applications in industry.
In a review article by Chi et al. (2009) the extracellular enzyme production, their properties and cloning of the genes encoding the enzymes from marine yeasts are overviewed. The extracellular enzymes include cellulose, alkaline protease, aspartic protease, amylase, inulinase, lipase, phytase and killer toxin. It was found that some properties of the enzymes from the marine yeasts are unique than that of the enzymes from terrestrial yeasts. Marine yeasts are versatile agents of biodegradation. They act on varied substrates and helps in nutrient recycling.
Chi et al., (2009) studied that the extracellular enzyme production, their properties and cloning of the genes encoding the enzymes from marine yeasts are described. The extra cellular enzymes include cellulase, alkaline protease, aspartic protease, amylase, inulinase, lipase, phytase and killer toxin. It was found that some properties of the enzymes from the marine yeasts are unique than that of the enzymes from terrestrial yeasts.
One hundred and nineteen amylases producing strains (29 yeasts and 90 bacteria) were isolated from some Cameroonian soils contaminated by starchy residues and screened for thermostable amylases production. Phenotypic characterization of these amylases producing strains revealed the prominence of ascomycetous yeasts and two kinds of bacteria, the aerobic endospore forming dominated by Bacillus sp and aero-anaerobic non spore forming bacteria dominated by lactic acid bacteria of Lactobacillus sp. Among yeasts, one designated 04LBA3 produced high title of very high thermostable amylase. It was able to provoke starch hydrolysis halo of 33.7±1.5 mm on starch agar plate, and produced 80 ±0.5 U/ml of amylase in starch broth after 48 h of incubation at 30°C. Concerning amylases producing bacteria, two isolates designated 04BBA15 and 04BBA19 showed very high amylolytic power, the values were 55.0±3.2 mm and 45.3±1.5 mm of starch hydrolysis halo respectively for 04BBA15 and 04BBA19. Amylase production in starch broth were 131.0 and 107.7 U/ml respectively for 04BBA15 and 04BBA19 after 48 h of incubation at 40°C, on the other hand, their crude amylase extract remained 100% of original activity after been heated at 80°C for 30 min. The strain 04LBA3, 04BBA15, 04BBA19 were respectively identified as strain of Schwanniomyces alluvius, Bacillus amyloliquefaciens and Lactobacillus fermentum (Fossi et al., 2009).
Several species isolated Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4°C and 20°C, indicating that they could be metabolically active in the sampled substrates (Vaz et al., 2011).
A total of 14 samples from different food sources viz., dahi, jaggery and different kinds of fruit juices were collected randomly from different localities of Kolkata, West Bengal, India. Thirty-three yeast strains were isolated using selective medium, Martin’s Rose Bengal Agar. Differential tests were applied including morphological, cultural and biochemical characteristics, which facilitate the opportunity for identification of the yeasts. The total number of isolated yeast strains was 12 from dahi, 2 each from apple juice, pineapple juice, mango juice, musambi juice, grape juice, orange juice, jaggery and 7 from sugarcane juice. These strains were found to produce various extra cellular enzymes and could ferment various carbon sources for the production of alcohol (Chatterjee et al., 2011).
A total of 245 yeast isolates from Gunung Halimun National Park (GHNP) were screened for cellulolytic activity using 0.2% cellulose-azure. The results showed that 16 isolates have cellulolytic activity using cellulose-azure assay. These isolates were further screened for carboxymethyl cellulase (CMCase), avicelase and cellobiase using specific substrates (carboxymethyl cellulosa, avicel and cellobiose) with Teather and Wood method. The seven isolates have CMCase; 6 isolates have cellobiase; 2 isolates have CMCase and cellobiase; and 1 isolate has CMCase and avicelase and cellobiase activities. Isolate S 4121 has the highest CMCase activity and identified as Trichosporon sporotrichoides (van Oorschot) van Oorschot and de Hoog UICC Y-286. (Mangunwardoyo et al., 2011).
One hundred forty-four microorganisms previously isolated from coffee fruit (Coffea arabica) were grown on casein agar to evaluate their proteolytic activities. Fifty percent of filamentous fungi, 52.5% of bacteria and 2.6% of yeasts were able to secrete proteases. Positive isolates were further examined in liquid culture for their protease activities by hydrolysis of casein at different pH values (5.0, 7.0 and 9.0) at 30 o C. Bacillus megaterium, B. subtilis, Enterobacter agglomerans, Kurthia sp, Pseudomonas paucimobilis and Tatumella ptyseos demonstrated the highest proteolytic activities at pH 9.0. One yeast isolate, Citeromyces matritensis, had a proteolytic activity of 2.40 U at pH 5.0. Aspergillus dimorphicus, A. ochraceus, Fusarium moniliforme, F. solani, Penicillium fellutanum and P. waksmanii showed the highest activities. Of the bacterial isolates, the highest enzyme activities were observed in B. subtilis 333 (27.1 U), Tatumella ptyseos (27.0 U) and B. megaterium 817 (26.2 U). Of the filamentous fungi, Aspergillus ochraceus (48.7 U), Fusarium moniliforme 221 (37.5 U) and F. solani 359 (37.4 U) had the highest activities at pH 9.0 respectively (Rodarte et al., 2011).
Zeni et al. (2011) studied that the screening of microorganisms, previously isolated from samples of agro-industrial waste and belonging to the culture collection of our laboratory, able to produce polygalacturonases (PG). A total of 107 microorganisms, 92 newly isolated and 15 pre-identified, were selected as potential producers of enzymes with PG activity. From these microorganisms, 20 strains were able to synthesize PG with activities above 3 UmL?1. After the kinetic study, the enzyme activity was increased up to 13 times and the microorganism identified as Aspergillus niger ATCC 9642 and the newly isolated W23, W43, and D2 (Penicillium sp.) after 24 h of fermentation led to PG activities of 30, 41, 43, and 45 UmL?1 , respectively.
Goldbeck et al. (2012) evaluated that the screening and identification of cellulase producing wild yeasts, isolated from samples collected from different Brazilian biomes. They were selected according to their capabilities of degrading carboxymethyl cellulose (CMC) and micro-crystalline cellulose (SERVACEL® ), as single carbon sources in solid medium. After the step of solid medium selection, yeast cells were grown in liquid medium containing cellulose (SERVACEL®); in shake flasks at temperature of 30°C and 150rpm agitation for 288 h. Three specific activities were evaluated: endoglucanase (CMCase), total activity (filter paper activity), and cellobiase. From a total of 390 strains of wild yeasts previously isolated, 16 strains performed cellulose hydrolysis, verified by the colorless halo in the solid medium. Among these 16 strains, 5 stood out as presenting higher levels of enzyme activity. The following step, screening in liquid medium, indicated only one strain as a potential producer of cellulases, named as AAJ6, for which the highest hydrolytic activity on carboxymethyl cellulose (0.33U/ml) and filter paper (0.039 U/ml) was recorded. Afterwards, this wild yeast strain (AAJ6) was molecularly identified by sequencing the ITS1-5.8S-ITS2 and D1/D2 domains of the subunit (26S) ribosomal DNA. Sequencing resulted in the identification of this strain as yeast-like fungus Acremonium strictum.
Enzymes are well known for their catalytic activities. Further it is seen that there are different sources of enzyme secretion and synthesis viz., bacteria, fungi, yeast, plant etc. Basically lipase enzyme has been used for industrial and research purposes because of its low cost and high stability. Lipase can be isolated from bacteria residing in oily soils. The isolated bacteria were identified and these belong to Staphylococcus class. Further screening was done by using tributyrin as substrate in nutrient agar media. Biochemical characterization along with motility tests were also performed for its identification. Statistical optimization of media parameters was done to analyze the enzyme activity in which effect of pH, incubation period, temperature, agitation speed and carbon and nitrogen source in form of oil and extract was carried out. It was observed that all the above said parameters affect the enzymatic activity of this lipolytic enzyme i.e lipase (Singh et al., 2013).
Five different sources were used for the isolation of the yeasts. For screening of the amylase production, the isolates were incubated in Amylase Activity Medium at 30°C for 3 days. The amylase activity was determined by dinitrosalicylic acid method. In total, twenty five yeast isolates were obtained from five different sources. Following the incubation in medium containing starch for the screening of amylase production, it was found that 12 yeast isolates produce amylase, and among the isolates, three of them showed the highest amylase activity. The isolates (19-3, 19-6 and 19-7) having the highest activity were identified as Saccharomycopsis fibuligera. The strains showed the highest amylase production at 30°C and pH 5.5. The favorable fermentation conditions and the selection of suitable growth parameters played key roles in the production of amylase by Saccharomycopsis fibuligera (Tansel Yalc?n and Cengiz Corbaci, 2013).
The hydrolytic potential of the marine yeast isolates obtained from the slope sediments of Arabian Sea and Bay of Bengal. The optimum growth conditions like temperature, salinity and pH of the isolates were also determined. The isolates from Bay of Bengal showed more enzyme production than those of Arabian Sea. All the isolates were lipolytic. Oxidative forms were more in abundance than the fermentative forms. Black yeasts obtained from the study area, showed maximum hydrolytic potential than compared to their counter parts. Majority of the isolates preferred 30°C, pH 6 and 15 ppt salinity for maximal growth (Kutty et al., 2014).
Marine yeasts are versatile agents of biodegradation. They act on varied substrates and helps in nutrient recycling. This study mainly focuses on the hydrolytic potential of the marine yeast isolates obtained from the slope sediments of Arabian Sea and Bay of Bengal. The optimum growth conditions like temperature, salinity and pH of the isolates were also determined. The isolates from Bay of Bengal showed more enzyme production than those of Arabian Sea. All the isolates were lipolytic. Oxidative forms were more in abundance than the fermentative forms. Black yeasts obtained from the study area, showed maximum hydrolytic potential than compared to their counter parts. Majority of the isolates preferred 30°C, pH 6 and 15 ppt salinity for maximal growth. (Kutty et al., 2014).
Kanzy et al. (2015) evaluated that the identified strain and the reference strain Rhodotorula glutinis were grown to study the effect of NaCl concentration, incubation temperature, initial pH and incubation period on dry biomass and enzyme production. The maximum biomass (13.95 g/l) and volumetric carotenoid production (6.544 mg/l) were scored by the reference strain R. glutinis after incubation for 120 hr at 30 °C and pH 6.6 in a medium containing 3% NaCl. the isolated strain showed its maximum biomass (9.02 g/l) in a medium containing 10% NaCl while the highest amount of enzyme (5.044 mg/l) were obtained in a medium containing 6% NaCl after 120 hr incubated at 30°C and pH 6.6. The obtained result showed that R. mucilagenosa will be a promising microorganism for commercial production of enzymes.
Proteases are important enzymes for various applications of industrial importance. Microbial proteases play an imperative task in various biotechnological processes. The endeavors of this work were to screen and isolate proteolytic microbes from soil. Banasthali (Tonk district) is situated in southeast Rajasthan. The geo-climatic location of Banasthali has been considered to be highly rich in wide variety of diversity of macro flora and fauna. Soil is the ideal habitat for many extracellular enzyme producing microorganisms. Bacterial and fungal isolates were screened for extracellular protease production. For qualitative analysis (zone of hydrolysis), all isolates were inoculated in gelatin agar plate and skim milk agar plate. Protease production was observed by the occurrence of clear zone around bacterial and fungal colonies. Three bacterial and three fungal isolates selected by initial screening were subjected to extracellular protease production in protease production medium and it was found that bacterial isolate (No. 2 from sample 1) was producing maximum protease activity (37.94 U/mL) after 3 days of incubation. Likewise fungal isolate (No. 3 from sample 1) was showing highest protease activity (51.33 U/mL) after 5 days of incubation (Sharma et al., 2015).
The amylase producing strain could also be helpful industrial application particularly in food and beverages, animal feed, brewing textiles, detergents and health care Shanmugasundaram et al. (2015).
Otero et al. (2015) studied that the 119 yeast strains were isolated and evaluated in terms of their ability to degrade xylan, which was found in the medium by using agar degradation halos, the basis of this polysaccharide, and Congo red dye. Selected microorganisms were grown in complex medium and the enzymatic activities of endo-xylanase, ?-xylosidase, carboxymetilcellulase, and filter paper cellulose were determined over 96 h of cultivation; the pH and biomass concentration were also evaluated. The yeast strain 18Y, which was isolated from chicory and later identified as Cryptococcus laurentii, showed the highest endo-xylanase activity (2.7 U.mL-1). This strain had the ability to produce xylanase with low levels of cellulase production (both CMCase 0.11 U.mL-1 and FPase 0.10 U.mL-1 recorded respectively.
Bessadok et al. (2015) studied that the isolation and identification of marine yeast strains from seawater, sediments seaweed, and fish/shrimp coproducts. Over six-different identified species of marine yeast, Yarrowia lipolytica strain having a proteolytic activity. Enzyme extracts showed that the relative optimal enzymatic activity was reached at pH = 9.0 and temperature of 45.0° C.
Premalatha et al. (2015) reported that the forty nine isolates of cellulose-degrading yeast were isolated from different sugar factories enriching by using the carboxymethyl cellulose agar medium (CMC). The cellulase activity of the organism diameter of clear zone around the colony and hydrolytic efficiency on cellulose were measured by Congo red assay. Among the yeast isolates CYLL21 and CYLL32 exhibited the maximum zone of clearance around the colony with diameter of 28 and 26 mm and the cellulolytic efficiency of 280 and 250 per cent respectively. The extracellular cellulase activity ranged from 22.0 to 17.83 U/ml in 48 h at 35°C for yeast isolates.
Twenty yeast strains were screened for pectinolytic activity, among the strains, only nine were positive for pectinase production. The best strain was Kluyveromyces marxianus NRRL-Y-1109 which gave high quantities of pectinase activity, by submerged fermentation. Different parameters such as incubation time, pH (3.5-7), temperature (20-45°C), nitrogen and carbon source, were optimized. The optimal incubation time, temperature and pH for pectinase production were found to be 48 hrs, 30°C and 6, respectively. Studies were conducted on the production of pectinase in submerged fermentation using agro-industrial residues such as wheat bran, grape waste, brewer’s malt, beet molasses and corncob at a concentration of 1.5% (wt./vol.). Under optimized fermentation conditions, maximal enzyme were produced when citrus pectin (4.8 U/mL) was used as the carbon source, but high enzyme production was also obtained on wheat bran (2.2 U/mL) and grape waste (1.8 U/mL) in shaking conditions (120 rpm) for 48 h. Peptone and yeast extract used together as nitrogen source gave best enzyme production. The effects of temperature (30-70°C), pH (3.5-8) and salt concentration (1, 2, 5 and 10%) on the pectinase activity were determined. The optimum activity was obtained when temperature 45°C and pH at 5.5. The enzyme was stable at 45°C (Oskay and Yalcin, 2015).
Carrasco et al. (2016) worked that the ability of yeasts isolated from the Antarctic region to grow on starch or carboxymethylcellulose and their potential extracellular amylases and cellulases. All tested yeasts were able to grow with soluble starch or carboxymethylcellulose as the sole carbon source; however, not all of them produced ethanol by fermentation of these carbon sources. For the majority of the yeast species, the extracellular amylase or cellulase activity was higher when cultured in medium supplemented with glucose rather than with soluble starch or carboxymethylcellulose. Additionally, higher amylase activities were observed when tested at pH 5.4 and 6.2, and at 30–37 °C, except for Rhodotorula glacialis that showed elevated activity at 10–22 °C. The cellulase activity was high until pH 6.2 and between 22–37 °C, while the sample from Mrakia blollopis showed high activity at 4–22 °C. Peptide mass fingerprinting analysis of a potential amylase from Tetracladium sp. of about 70 kDa, showed several peptides with positive matches with glucoamylases from other fungi.
Aspartic proteases are of significant importance for medicine and biotechnology. In spite of sufficient evidence that many non-Saccharomyces yeasts produce extracellular proteases, previous research has focused on the enzymes of Candida species because of their role as virulence factors. Nowadays, there is also increasing interest for their applications in industrial processes, mainly because of their activities at low pH values. Here, we report the features of new acid proteases isolated from wine-relevant yeasts Metschnikovia pulcherrima and Wickerhamomyces anomalus. This is the first detailed description of such an enzyme derived from strains of W. anomalus. Deviating to most former studies, we could demonstrate that the yeasts produce these enzymes in a natural substrate (grape juice) during the active growth phase. The enzymes were purified from concentrated grape juice by preparative isoelectric focusing. Biochemical data (maximum activity at ? pH 3.0, inhibition by pepstatin A) classify them as aspartic proteases. For W. anomalus 227, this assumption was confirmed by the protein sequence of WaAPR1 determined by LC-MS/MS. The sequence revealed a signal peptide for secretion, as well as a peptidase A1 domain with two aspartate residues in the active site. The enzyme has a calculated molecular mass of 47 kDa and an isolelectric point of 4.11. (Schlander et al., 2017).
Poonam and Nivedita (2018) evaluated that the screening pectinase producing bacteria from Rhizoclonium sp. algal biomass collected from different parts of Himachal Pradesh. Various optimization steps were carried out to make the production of pectinase enzyme cost effective and commercially viable. The various parameters studied for pectinase production were different media, medium pH, temperature, inoculum size and incubation period. The maximum pectinase production was observed at initial pH of 5.0, at 30°C with 10% inoculum size followed by incubation period of 72 hrs.
The species identification intra-specific variation of yeasts isolated from Handia was studied by restriction digestion of their 5.8S ITS region between 18S rRNA and 26S rRNA genes amplified by PCR with ITS 1 and ITS 4 primers. The PCR amplified product for all four strains of S. cerevisiae was 880 bp which is in agreement with those previously reported for yeast strains (Clemente-Jimenez et al., 2004; Pulvirenti et al., 2001).
Restriction analysis of the PCR amplified product of the 5.8S-ITS region of all four S. cerevisiae with HaeIII, HinfI and PstI generated a restriction fragment pattern consisting of four fragments with HaeIII (320, 240, 180 and 140 bp) and three fragments with HinfI (370, 370 and 130 bp). The PCR product 5.8S-ITS region of S. cerevisiae remained uncut after PstI digestion. Similar banding patterns with HaeIII and HinfI were also observed for the reference strains of S. cerevisiae MTCC 180, S. cerevisiae MTCC 178 and S. cerevisiae MTCC 211 and the reported strains (Esteve-Zarzoso et al., 1999; Jeyarama et al., 2008). Although this technique could not differentiate the four isolates of S. cerevisiae at strain level, their identity to species level was confirmed.
The PCR product remained uncut after digestion with HaeIII and PstI. The previous work also demonstrated that the PCR product of ITS-5.8S rDNA of reported strain of H. guilliermondii remained uncut with HaeIII. The PCR amplification of 5.8S ITS region of the genomic DNA of H. guilliermondii G4 yielded 775 bp fragments, similar to the results reported previously (Esteve-Zarzoso et al., 2001). The restriction digestion of the 5.8S-ITS region of the PCR amplified product of H. guilliermondii G4 with HinfI produced approximately 385, 200, 160 and 100 bp for the identification of G4 as H. guilliermondii. (Esteve-Zarzoso et al., 2001).
The PCR amplification of 5.8S ITS region of the genomic DNA of both P. kudriavzevii H21L and P. kudriavzevii MTCC642 yielded 500 bp fragment, similar to the results reported previously (Latorre-Garcia et al., 2007). Digestion of this product from P. kudriavzevii H21L with HaeIII yielded two fragments of approximately 380 and 100 bp whereas HinfI produced fragments of 250 and 150 bp. The strain P. kudriavzevii MTCC 642 displayed a restriction pattern consisting of approximately 320 and 100 bp with HaeIII and 280 and 130 bp fragments with HinfI. The PCR products of both the P. kudriavzevii remained uncut after digestion with PstI. Although the 5.8S-ITS pattern of P. kudriavzevii H21L generated with HaeIII was found to be similar to published strains (EL-Sharoud et al., 2009), it differed from that of P. kudriavzevii MTCC 642.
Amplification of ITS2/ 5.8S rDNA was proved as a potential and rapid technique for identification of fungi causing corneal infection by Ferrer et al. (2001). Fifty strains of 12 fungal species (yeasts and molds) were evaluated by PCR amplification method, amplified DNA was sequenced, aligned against sequences in GenBank. Molecular identification of fungi was successful in all the cases, thus proved the amplification of ITS2 and 5.8S rDNA as a potential and rapid technique for identification of fungi.
C. glabrata has been proven to be more closely related to S. cerevisiae than C. albicans, based on 18S rRNA sequence homology studies (Barns et al., 1991). C. glabrata has been evolved after an event of whole genome duplication (WGD) followed by increased rate of gene loss as compared to S. cerevisiae, both of them share a common ancestor (Dujon et al., 2004).
Most of C. glabrata genes are orthologous to S. cerevisiae genes due to their origin from same ancestor (Dujon et al., 2004; Marcet-Houben and Gabaldon, 2009). Despite of the evolutionary relatedness, C. glabrata is asexual haploid yeast while S. cerevisiae can switch between haploid and diploid forms and exhibit sexual life cycle (Wong et al., 2002). Earlier C. glabrata was named Cryptococcus glabratus due to its ability to form psuedohyphae in nitrogen starving medium and then Torulopsis glabrata but after few more studies on it, it has given place in Candida genus (Bialkova and Subik, 2006).
Yeasts antagonistic to Colletotrichum capsici were isolated from Thai fruits and vegetables. Four antagonists (R13, R6, ER1, and L2) were found that inhibited C. capsici growth with biocontrol efficacies of 93.3%, 83.1%, 76.6%, and 66.4% respectively. Identification by 26S rDNA, and ITS region sequence together with physiological and morphological characteristics, showed them to be Pichia guilliermondii, Candida musae, Issatchenkia orientalis and Candida quercitrusa, in order of their efficacy. P. guilliermondii strain R13 showed efficacy in reducing disease incidence on C. capsici infected chilli fruits to as low as 6.5%. Lower disease incidence was observed at lower storage temperature (Chanchaichaovivat et al., 2007).
Twelve seawater samples were collected from the coastal marine waters of northeastern Taiwan, and 109 yeast cultures were isolated from the samples. Isolates were first classified by phenotype and then into 9 groups according to the sequencing of 5.8S-ITS ribosomal DNA. The results showed that Candida tropicalis was the most frequently recovered yeast found in the coastal waters of northeastern Taiwan. Other species found in this study included C. glabrata, Saccharomyces yakushimaensis, Kazachstania jiainicus, Kodamaea ohmeri, Pichia anomala, Issatchenkia orientalis, and Hanseniaspora uvarum. The biodiversity of yeast species was determined from the south to north of the northeastern coastal waters (Yi-Sheng Chen, 2009).
Twelve seawater samples were collected from the coastal marine waters of northeastern Taiwan, and 109 yeast cultures were isolated from the samples. Isolates were first classified by phenotype and then into 9 groups according to restriction fragment length polymorphism (RFLP) analysis and the sequencing of 5.8S-ITS ribosomal DNA. The Candida tropicalis was the most frequently recovered yeast found in the coastal waters of northeastern Taiwan. Other species found in this study included C. glabrata, Saccharomyces yakushimaensis, Kazachstania jiainicus, Kodamaea ohmeri, Pichia anomala, Issatchenkia orientalis and Hanseniaspora uvarum. The biodiversity of yeast species was determined from the south to north of the northeastern coastal waters. (Chen et al., 2009).
The diversity of culturable yeasts at deep sea hydrothermal sites have suggested possible interactions with endemic fauna. Samples were collected during various oceanographic cruises at the Mid-Atlantic Ridge, South Pacific Basins and East Pacific Rise. Cultures of 32 isolates, mostly associated with animals, were collected. Phylogenetic analyses of 26S rRNA gene sequences revealed that the yeasts belonged to Ascomycota and Basidiomycota phyla, with the identification of several genera: Rhodotorula, Rhodosporidium, Candida, Debaryomyces and Cryptococcus. These genera are usually isolated from deep-sea environments (Burgaud et al., 2010).
The yeast microbiota is isolated from soil and water samples collected on King George Island. A high number of yeast isolates was obtained from 34 soil and 14 water samples. Molecular analyses based on rDNA sequences revealed 22 yeast species belonging to 12 genera, with Mrakia and Cryptococcus genera containing the highest species diversity. The species Sporidiobolus salmonicolor was by far the most ubiquitous, being identified in 24 isolates from 13 different samples. Most of the yeasts were psychrotolerant and ranged widely in their ability to assimilate carbon sources (consuming from 1 to 27 of the 29 carbon sources tested). All species displayed at least 1 of the 8 extracellular enzyme activities tested. Lipase, amylase and esterase activity dominated, while chitinase and xylanase were less common. Two yeasts identified as Leuconeurospora sp. and Dioszegia fristingensis displayed 6 enzyme activities (Mario Carrasco et al., 2012).
Hashem et al., (2013) evaluated that the two yeast strains were isolated and identified by sequencing of ITS1 and ITS2 regions. Comparing the sequence results with the GenBank reference proved that the strain Kluyveromyces sp. ZMS1 had 100% of similarity with Kluyveromyces maxianus. Yeast strain Kluyveromyces sp. ZMS3 had only 97% of similarity with the reference species; consequently, it could be a new strain. At 35°C, Kluyveromyces sp. ZMS1 GU133329 and Kluyveromyces sp. ZMS3 GU133331 produced 9.55 (w/v) and 11.72% (w/v) of ethanol, respectively. The appropriate concentration of sugar that induced the maximum production of ethanol by these strains was 20 to 25%. The optimum pH range for both strains was 5.0-5.5. In fed-batch culture, the maximum ethanol production was 11.71 (w/v) and 11.62% (w/v) by Kluyveromyces sp. ZMS3 GU133331 and Kluyveromyces sp. ZMS1 GU133329 respectively.
Karimi and Hassanshahian (2016) studied that the phenol-degrading yeast from environmental samples (soil and wastewater) was isolated from the coking plant of Zarand, Kerman. Eleven yeasts were isolated and identified the phenol effluent soil samples. These phenol degrading yeasts were identified by molecular method using amplification of 18S rRNA gene region. The sequencing results showed that these isolated yeasts belonged to Candida tropicalis strain K1, Pichia guilliermondii strain K2, Meyerozyma guilliermondii strain K7 and C. tropicalis strain K11 respectively.
Nicole DeLong and Les Erickson (2017) examined that the isolation, identification and characterization of wild yeasts from the local environment and to make them available to both commercial and craft brewers for making novel beers. The use of locally-sourced yeasts could also be used by commercial brewers as a marketing angle, as is often done with local hops, grains, or water. As a source of wild yeasts, we obtained various wild and cultivated fruits growing in rural Maryland and a sample of Belgium-style ale being aged in used oak wine barrels at Evolution Craft Brewing Company (Salisbury, MD). Yeast colonies were isolated using yeast extract/peptone/dextrose (YPD) agar plates containing antibiotics and identified by colony morphology as well as PCR amplification and sequencing of the 5.8S rRNA genes.
Mishra et al. (2018) studied that the six yeast cultures were isolated from twenty indigenous fermented food samples collected from various regions of Meghalaya. Isolation was conducted on specific media: MA, SCA, RBA and YPDA for yeast isolates. Based on the phenotypic characteristics obtained from Gram’s staining, biochemical characterization of selected isolates was accomplished by API 20 C AUX V5.0 kit for yeast followed by 5.8s rRNA amplification for its genotypic identification and the sequences was deposited at Genebank and NCBI bearing their specific accession numbers were obtained.
MATERIALS AND METHODS
3.1 Study sites
Muthupet is a panchayat town in Thiruvarur district in the Indian state of Tamil Nadu. Muthupet is also known as Pearlpet. Muthupet comes under the Thiruthuraipoondi assembly constituency which elects a member to the Tamil Nadu Legislative Assembly once every five years and it is a part of the Nagapattinam (Lok Sabha constituency) which elects its Member of Parliament (MP) once in five years. The town is administered by the Muthupet town panchayat. In the present study four study area of Manakkattai, Saviyarmunai, Vauvalthottam and Deveraraul in Thiruvarur Dist were chosen (Fig. 1).
Fig. 1: Study area
3.2 Sample collection
Seawater soil samples were collected different sites from east coast of mangrove environments of Thiruvarur District, Tamilnadu. Sea water soil samples (from 10 to 15 cm) were collected at each station by walking along the shoreline. Each sample was collected in a sterilized bottle, which was rinsed twice with the sea water before use. The collected water and soil samples were brought to the laboratory in sterilized polythene bags or sterilized bottle, handpicked, air dried and stored in containers for future use.
3.3 Isolation of marine yeast (Wickerham, 1951)
For isolation of yeast from the collected samples, dilution plate technique was used. The soil and water samples was weighed and serially diluted up to 10-5. Then serial dilutions were prepared. The dilution was done on board employing spread plate method. To prepare malt-yeast-glucose-peptone agar medium and supplemented with 200mg/l chloramphenicol, then pour the plate for undisturbed condition. After solidification, 0.1 ml of the 10-3, 10-4 and 10-5 dilution was spread plated on malt-yeast-glucose-peptone agar supplemented with 200 mg/l chloramphenicol. The plates were incubated for 5-7 days at 25±1oC. After the incubation period, the yeast colonies were observed with the colony forming unit (CFU). The colonies developed were purified by quadrant streaking and transferred to malt extract agar slants for further studies.
3.4 Morphological and micrographic investigation
The colonies were observed and described on malt yeast glucose peptone agar medium. The isolates were grown in Malt Yeast Glucose Peptone broth for determination of these cultural characteristics (pellicle, sedimentation or ring formation, colony of colony, elevation of colony, texture of colony and shape of colony) under microscope.
Yeast isolates purified by quadrant streaking in malt extract agar were preserved in vials overlaid with sterile liquid paraffin.
3.4.2 Identification of the Isolates
The isolated yeast strains were identified up to genera as per Barnett et al. (1990). For this, microscopic appearance of the cell, mode of reproduction and certain biochemical and physiological characteristics were studied.
188.8.131.52 Microscopic appearance of yeast cells
a) Vegetative cells:
Young growing yeast cultures were inoculated into sterile malt extract broth and incubated at 28 ± 2 oC for 48 hrs. Wet mount preparations of the cultures were observed under oil immersion microscope for the following characteristics: a) Whether the yeast reproduce by budding, splitting or both, b) The shape and size of the vegetative cells, c) If the yeast form buds where do they occur on the mother cell.
b) Microscopic examination for filamentous growth
Slide cultures of isolated yeasts were prepared. For this, malt extract agar plates were prepared. In each plate four sterile cover slips dipped in malt extract agar (1 % agar) was kept on the medium surface at 45o angle position by gently piercing the agar. These slides were examined microscopically daily or once in two days for up to about 2 weeks. Observations were done to ascertain whether or not there is filamentous growth. If so, what kind of cells grow from filaments, can be observed.
3.5 Analysis of physicochemical parameters in water samples
Follows the instructions given by manufacture to use the pH meter. Essential aspect to use all the pH meters to calibrate it with suitable buffers. Ready buffer of pH values are also available in the market, set the pH meter with a buffer whose value is near to the expected pH of the sample. pH of water samples were measured by immersing the glass electrodes in sample solution under study.
Temperature measurement is made by taking a portion of the water sample (about 1litre) and immersing the thermometer into it for a sufficient period of time (till the reading stabilizes) and the reading is taken, expressed as °C.
Transparency is measured by gradually lowering the Secchi disc at respective sampling points. The depth at which it disappears in the water (X1) and reappears (X2) is noted. The transparency of the water body is computed as follows:
Transparency (Secchi Disc Transparency) = (X1+ X2)/2
Where, X1 = Depth at which Secchi disc disappears
X2 = Depth at which Secchi disc reappears
3.5.4 Electrical conductivity
Electrical conductivity was measured by Systronic Direct Reading Conductivity Meter (model 304). The results have been expressed in mohs/cm. The conductivity was measured at room temperature.
Electrical conductivity =observed conductivity x cell constant x temperature factor at 298k.
3.5.5 Hardness (EDTA method)
Take 50ml water sample in a conical flask. If sample is having higher Calcium, take a smaller volume and dilute to 50ml. Add 1ml of buffer solution. If the sample is having higher amount of heavy metals add 1ml of Na2S solutions. Add 100-200 mg Eriochrome Block T Indicator in the Solution to turn wine red. Titrate the contents against 0.01, M EDTA solution. At the end point, color changes from wine red to blue.
Hardness as mg/L CaCO3 = ml of EDTA used x 1000 ml of sample taken
3.5.6 Total Alkalinity, Bicarbonate
Total alkalinity of the water sample was measured in terms of volume of sample required to neutralize the strong acid. HCl, using phenolphthalein as indicator and further with methyl orange. The alkalinity by using Phenolopthaline indicator is called ”Phenolopthaline” alkalinity and is indicated as PA. Alkalinity by using methyl orange is called Total alkalinity and is indicated as TA. To determine alkalinity, method given in the literature. (APHA- AWWA, 1995) was followed and PA. TA values were calculated by using following formula.
PA as CaCO3 (mg/L) = A x N. HCl x 1000 x 50 Vol. of Sample
TA as CaCO3 (mg/L) = B x N. HCl x 1000 x 50 Vol. of Sample taken
A = ml of HCl required with Phenolphthalein only
B = ml of total HCl required with Phenolphthalein and methyl orange
3.5.7 Total dissolved solids
Dissolved solids are solids that are in dissolved state in solution. Waters with high dissolved solids generally are of inferior palatability and may induce an unfavourable physiological reaction in the transient consumer.
The difference in the weights of Total Solids (W1) and Total Suspended Solids (W2) expressed in the same units gives Total Dissolved Solids (TDS).
(W1-W2) X 1000
W1 = Weight of total solids + dish
W2 = Weight of total suspended solids
A known volume (50ml) of the sample is pipette into a porcelain dish and evaporated to dryness on a hot water bath. 2ml of phenol disulphonic acid is added to dissolve the residue by constant stirring with a glass rod. Concentrated solution of sodium hydroxide or conc. ammonium hydroxide and distilled water is added with stirring to make it alkaline. This is filtered into a Nessler’s tube and made up to 50ml with distilled water. The absorbance is read at 410nm using a spectrophotometer after the development of colour. The standard graph is plotted by taking concentration along X-axis and the spectrophotometric readings (absorbance) along Y-axis. The value of nitrate is found by comparing absorbance of sample with the standard curve and expressed in mg/L.
3.5.9 Orthophosphates and sulphate
100ml of the sample is filtered into a Nessler’s tube containing 5ml of conditioning reagent. About 0.2g of barium chloride crystals is added with continued stirring. A working standard is prepared by taking 1ml of the standard, 5ml of conditioning reagent and made up to 100ml, to give 100 NTU. The turbidity developed by the sample and the standards are measured using a Nephelometer and the results are tabulated.
3.5.10 Dissolved oxygen
The samples are collected in BOD bottles, to which 2ml of manganous sulphate and 2ml of potassium iodide are added and sealed. This is mixed well and the precipitate allowed settling down. At this stage 2 ml of conc. sulphuric acid is added, and mixed well until all the precipitate dissolves. 203 ml of the sample is measured into the conical flask and titrated against 0.025N sodium thiosulphate using starch as an indicator. The end point is the change of colour from blue to colourless.
3.5.11 Nitrite determination
Aliquots of stock solution containing 0.2 – 8.0 µgml-1 of nitrite were transferred in to series of 10 mL calibrated flask. To each flask, 1 ml of 0.5% sulfanilic acid and 1 mL of 2 moll-1 hydrochloric acid solutions were added and the solution was shaken thoroughly for 5 min to allow the diazotization reaction to go to completion. Then, 1 ml of 0.5% methyl anthranilate and 2 ml of 2 mol l-1 sodium hydroxide solutions were added to form an azo dye and the contents were diluted to 10 ml using water. After dilution to 10 ml with water, absorbance of the red colored dye was measured at 493 nm against the corresponding reagent blank and the calibration graph was constructed.
Take 250 ml of sample in a Kjeldahl flask. Add 15 ml borate buffer and 6N sodium hydroxide (Add NaOH until pH 9.5 is reached). Place 25 ml of boric acid solution containing 2-3 drops of mixed indicator n a conical flask below the condenser so that the tip of outlet of the condenser is dipped in contents of conical flask. Remove the conical flask containing distillate after distillation, which turns blue for dissolution of ammonia. Titrate the distillage against 0.01N hydrochloric acid until blue color changes to pink. Run a blank with distilled water in a similar way.
3.5.13 Determination of iron, manganese, zinc
100ml volume of water sample containing known anounts of elements (25µg of Fe, 10 µh of Mn and 5µg of Zn) was adjusted to the desired pH with hydrochloric acid or ammonia solutions. Then the required volume of chealting agents (2 ml 0.01 mol-1 EDTA for Fe and 2 ml of 0.01 mol-1 1,10 phenanthroline for Mn and Zn) was added to inform the metal chelae. The column was preconditioned with a solution having the optimum pH and the sample solution was passed through the column at a flow rate of about 1 ml min-1. The adsorbed metals as their chelates on the Ambersorb 572 were then eluted with 5 ml of 2 mol/l hydrochloric acid solution for Fe, and 5 ml of 2 mol/l nitric acid solution for Mn and Zn. The eluting was collected in a 5 ml volumetic flask and the metals were determined by FAAS method.
3.6 Analysis of physico-chemical properties of soil samples
To determine the pH at the moisture saturation percentage of soil, take 50 gm of 2.4 mm soil (pass through 2.4 mm pore sieve) in beaker. Add small increment of distilled water without stirring, till glistering layer appears on the surface of the soil. Now stir the soil with the help of a glass rod to make uniform paste. If pH to be determined in 1:5 soil solution, take 20 gm soil and add 100 ml of distilled water to it. Stir for about an hour at regular interval. Determine the pH electrometrically using glass electrode pH meter, as described for the analysis of water. Take the pH of unfiltered soil suspension. Calculations express the results directly in pH in specifying the dilution of the soil suspension.
3.6.2 Electrical Conductivity
From 1:5 soil solutions as mentioned in method of measurement of pH, the electrical conductivity was measured within an hour by direct reading conductivity meter. The temperature of soil suspension was noted and the conductivity at 25°C was calculated as below. Conductivity at 25°C = observed conductivity x cell constant x Temperature factor at 298k. The results are expressed as conductivity in mohs/cm at 25°C in 1:5 soil suspensions
3.6.3 Estimation of organic carbon
The determination of soil organic carbon was estimated based on the chromic acid wet oxidation method. 10 – 20 mg soil was weighed into a dry tarred 250 ml conical flask to which 10 ml (1N) K2Cr2O7 was added and swirled gently to disperse the soil in the solution. Then 20 ml concentrated H2SO4 was added and immediately the flask was swirled until the soil and the reagents were mixed and heated while swirling the flask on a hot plate until the temperature reached 135°C (approximately ½ a minute). It was then set aside to cool slowly on an asbestos sheet in a fume cupboard. Two blanks (without soil) also were running in the same way to standardise the FeSO4 solution. When cooled (20 – 30 minutes), diluted to 200 ml with deionised water and proceeded with the FeSO4 titration using the “ferroin” indicator. Three or four drops of ferroin indicator was added and titrated with 0.4 N FeSO4. As the end point was approached, the solution took on a greenish colour and then changed to a dark green. At this point, ferrous sulphate was added drop-by-drop until the colour changed sharply from blue-green to reddish-grey (Allison and Black, 1965).
The percentage carbon was determined from the following formula:
Organic carbon (%) =0.003 g x N x 10 ml x (1 T/S) ODW×1003(1- T/S) WWhere: N = Normality of K2Cr2O7 solution T = Volume of FeSO4 used in sample titration (ml) S = Volume of FeSO4 used in blank titration (ml) ODW = Oven-dry sample weight (g).
3.6.4 Estimation of organic matter
In a dry conical flask 0.2 g of soil sample was taken. 2 ml of K2Cr2O7 followed by 4 ml concentrated H2SO4 were added to it and the contents were gently mixed. The flask was kept aside for 30 min for the complete reaction to take place and the contents were diluted by adding 40 ml marine water. 2 ml of phosphoric acid was added to it followed by the addition of 1 ml of diphenylamine indictor. The contents were titrated against 0.4 N ferrous ammonium sulfate until the colour changed to brilliant green. The blank titration was carried out with the same amounts of reagents but without the soil sample
Percentage of carbon (%) = 3.951(1-T / S) W 2.
Percentage of organic matter (%) = % of C 1.724
Here, W = Weight of soil in g T = ml of FAS S = Ferrous solution with blank titration (ml)
3.6.5 Estimation of available nitrogen
About 0.2 ml to 1 ml of working standard solution was pipetted out in S1 to S5 test tubes respectively. 0.2 ml of sample was taken in U1 test tube. All the test tubes were made up to 9 ml using sterile distilled water. 1.5 ml of sodium hydroxide and 1ml Nessler?s reagent was added to all tubes. The intensity of the colour development was read at 540 nm using green filter. The concentration of nitrogen of the solution was calculated using standard graph.
Test OD / test OD conc. of Std. 100/ volume of sample taken
3.6.6 Estimation of total phosphorous
In a china dish 0.5 g powdered soil was taken and it was moistened with distilled water to the consistency of a thin paste. 2 ml Conc. HNO3 followed by 2 ml Conc. Perchloric acid were added. The contents were heated slowly on a hot plate until they become nearly dry. The dish was cooled and 1 ml perchloric acid was added and the contents were treated again until they were dry. The dish was cooled and 20 ml of diluted H2SO4 was added. It was boiled slowly for 10 minutes and allowed to cool. The contents were filtered through Whatman no. 42 filter paper and the final volume was made up to 250 ml. 50 ml aliquot was taken in a beaker. 2 ml of ammonium molybdate and 5 drops of SnCl2 were added to it. After the development of colour, the absorbance was read in a spectrophotometer at 690 nm.
3.6.7 Calcium and Magnesium
50gm of soil was mixed with 100ml of 40% alcohol, shaken for 15 min and filtered by Whatman no 50 filter paper. The procedure was repeated 4 to 5 times. Finally soil was cleaned by absolute alcohol and was dried. Dry soil was scraped and mixed with 100ml sodium acetate solution. The mixture was stirred and placed overnight. It was filtered by Whatman no 42 filter paper and procedure was repeated four to five times. The filtrate was made to 500ml with distilled water volumetric flask. The Ammonium Acetate was evaporated and the remaining residue was dissolved in small amount of aquaregia. It was again evaporated to dryness and dissolved to 500ml solution in volumetric flask. Calcium and Magnesium containts from this solution were estimated by titration with EDTA using mureside and Erichrome Black T indicator.
3.6.8 Sodium and Potassium
Sodium and Potassium contents were estimated by flame photometer by using the solution left over after Calcium and Magnesium determination. For Sodium the filter used was of 589 nm-wave length and for potassium of 768 nm wave length.
3.6.9 Estimation of zinc, copper, iron and manganese
These procedures 1.97 g of diethylene triamine pentaacetic acid (DTPA) and 1.1 g calcium chloride (CaCl2) was weighed in a beaker. Dissolve with distilled water and then transfer to a 1-litre volumetric flask. In another beaker, 14.92 g of triethanolamine (TEA) is weighed and dissolved with distilled water and make up to 1 litre. The pH was adjusted to exactly 7.3 with 6 N hydrochloric acid (HCl), and make to 1-litre volume with distilled water. The final extractant solution is 0.005 M DTPA, 0.1 M TEA, 0.1 M CaCl2. A series of standard solutions for micronutrients in DTPA extraction solution was prepared. Fe: 0, 1, 2, 3, 4, 5 ppm; Zn: 0, 0.2, 0.4, 0.6, 0.8, 1.0 ppm; Cu: 0, 1, 2, 3, 4 ppm; Mn: 0, 1.0, 1.5, 2.0, 2.5 ppm. 10 g of air-dried sieved soil (2-mm) was weighed into a 125-ml Erlenmeyer flask. 20 ml of extractant solution was added. It was shaken for 2 hrs on a reciprocal shaker. The suspension was filtered through a Whatman no. 42 filter paper. Zn, Fe, Cu, and Mn were measured directly in the filtrate by an Atomic Absorption Spectrophotometer.
3.7 Diversity Indices
Diversity index is a mathematical measure of species diversity in a given community. Based on the species richness (number of species present) and species abundance (number of individuals). However, there are two types of indices, dominance indices and information statistic indices. The equations for the two indices:
Shannon Index H=-i=1SpilnpiSimpson Index D= 1i=1spi2,
Pielou’s Evenness Index e=H/logs.
The Shannon index is an information statistic index which means it assumes all species are represented in a sample and they are randomly sampled. In the Shannon index, p is the proportion (n/N) of individuals of one particular species found (n) divided by the total number of individuals found (N), ln is the natural log, ? is the sum of the calculations and s is the number of species (Simpson, 1949).
The Simpson index is a dominance index because it gives more weight to common or dominant species. In the Simpson index, p is the proportion (n/N) of individuals of one particular species found (n) divided by the total number of individuals found (N), ? is still the sum of the calculations, and S is the number of species. S (number of species) N (total number of individuals) ? (sum) of pi2 (n/N) 2 ? (sum) of pi ln pi (Shannon and Weaver 1949).
Species evenness was expressed by J=H’/H’max, where H’max is the maximum value of diversity for the number of species present (Pielou, 1975).
Screening of enzyme activity of yeast strain
3.8 Screening of enzymes from yeast isolates
3.8.1 Amlyase activity (Fossi et al., 2009)
Amylase producing yeast were screened on Amylase activity medium (AAM) (starch 5g/L; peptone 5g/L, yeast extract 5g/L, MgSO4.7H2O 0.5g/L, FeSO4. 7H2O 0.01g/L; NaCL 0.01 g/L, agar 15g/L) plates. Incubation at 30°C was carried out for 3 days, after which the plates were stained with lugol solution. The colonies forming the largest halo zone were observed.
3.8.2 Protease activity (Strauss et al. 2001)
Extracellular protease production was determined on YEPG medium containing 20 (g /l) 1 casein, pH 6. A clear zone around the colony indicated that protease activity was conformed.
3.8.3 Pectinase activity (Ankin and Anagnostakis, 1975)
The secretion of extracellular pectic enzymes was tested on the following medium (g/l) (Pectin -5g, yeast extract-1g, agar- 15g pH 5.0 in 1L distilled water). After cell growth, plates were flooded with hexadecyl trimethyl ammonium bromide (10 g /l).A clear halo around a colony in an otherwise opaque medium indicated degradation of the pectin.
3.8.4 Cellulase activity
Cellulose agar (casein hydrolysate 0.05 g; yeast extract 0.05 g; NaNO3 0.1 g; cellulose powder 0.5 g; agar 2 g; sea water 100 ml; pH 7) was used for testing cellulase production. The plates were spot inoculated and incubated at room temperature (28±2ºC) for 7 to 10 days. The zone of clearance around the colonies was noted as positive.
3.8.5 Esterase activity (Slifkin 2000)
The ability to hydrolyse esters was tested on the following medium (g l)1): peptone, 10; NaCl, 5; CaCl2.2H2O, 0Æ1; Tween 80 (polyoxyethylen-sorbitanmonooleate), 10 and agar, 20, pH 6-8. The presence of esterase activity (EsA) was seen as a visible precipitate (opaque halo) around the colony.
3.9 Production of the enzymes
The enzyme was produced, in the medium containing 1% starch and 1% yeast extract and inoculated in yeast culture, then cultivated for 3 days at room temperature. After removal of cells by centrifugation at 4oC, the supernatant was used as crude extract of enzyme which was used for hydrolyzing raw starch.
3.9.1 Amylase activity assay (Fuwa, 1951)
100 µL of enzyme solution was added to 100 µL 0.1% soluble starch solution, and incubated for 10 minutes in 50°C. Reaction was stopped by addition of 100 µL HCl 0.1 N. To visualize remaining starch in solution, 100 µL iodine solutions was added and the total volume brought to 2 mL by water addition. The absorbance was recorded on 600 nm wavelength. To calculate unit activity of the enzyme, following equation was used
Where : AU = Activity Units AC = Absorbance Control AS = Absorbance Sample Ve = Volume of enzyme used in assay f = dilution factor.
3.9.2 Protease activity assay (Kole et al., 1988)
The protease activity was assayed in duplicate with cell-free culture supernatants, using azocasein as the substrate. Enzymatic hydrolysis of azocasein produces stable dye-labelled peptides and amino acids into the reaction mixture which can be measured easily. Azocasein protease activity was measured by incubating 1 ml of culture supernatant and 1 ml of 0.5% (w/v) azocasein (Sigma) in 0.2 M Tris-hydroxymethyl amino methane hydrochloride (Tris-HCl) buffer (pH 7.4) in an incubator (Innova, New Brunswick Scientific) at 75°C for one hour. The reaction was stopped by adding 2 ml of 10% (w/v) trichloroacetic acid. The test tubes were allowed to stand for 30 min at room temperature. The mixture was thoroughly mixed using a vortex mixer (VF2, Jankel and Keunkel Kika Larbotechnik) before being centrifuged at 3 000 rpm for 10 min to remove a yellow precipitate. The absorbance of the supernatant was measured at 440 nm using a Shimadzu UV-120-2 spectrophotometer. The activity of the protease was expressed in arbitrary units, where 1 unit of activity is equivalent to change in optical density of 0.01 nm per min at 440 nm. The enzyme assays were done in duplicate for each sample.
3.9.3 Cellulase activity (Ghosh, 1987)
Activity of Cellulase in the culture filtrates was determined and quantified by carboxy-methyl cellulase method. The reaction mixture with 1.0 ml of 1% carboxymethyl cellulose in 0.2 M acetate buffer (pH 5.0) was pre-incubated at 50°C in a water bath for 20 minutes. An aliquot of 0.5 ml of culture filtrate with appropriate dilution was added to the reaction mixture and incubated at 50 °C in water bath for one hrs. Appropriate control without enzyme was simultaneously run. The reducing sugar produced in the reaction mixture was determined by dinitro- salicylic acid (DNS) method (Miller 1959). 3, 5-dinitro-salicylic acid reagent was added to aliquots of the reaction mixture and the color developed was read at wavelength 540 nm.
3.10 Qualitative analysis of bioactive compounds
Preliminary bioactive compounds was carried out for the extract as per standard methods described by Harbone, 1985.
3.10.1 Detection of alkaloids
Extracts were dissolved individually in dilute hydrochloric acid and filtered. The filtrates were used to test the presence of alkaloids.
3.10.2 Detection of flavonoids
Two ml of extract was taken a 10 ml test tube and added few drops of 1% ammonia solution. Change the yellow colouration was observed indicating the presence of flavonoids.
3.10.3 Detection of steroids
Two ml of acetic anhydride was added to five g of the plant extracts, each with two ml of H2SO2. The colour was changed from violet to blue or green in some samples indicate that the presence of steroids.
3.10.3 Detection of terpenoids
Five g of the extract of the stems was mixed with two ml of chloroform and concentrated H2SO2 (3ml) was carefully added to form a layer. An appearance of reddish brown colour in the inner face was indicates that the presence of terpenoids.
3.10.4 Detection of Phenols
Ten g extracts were treated with few drops of ferric chloride solution. Formation of bluish black colour indicates that the presence of phenol.
3.10.5 Detection of Saponins
2ml of extract was added in 5ml of distilled water in a water bath next shaken in vigorously for a stable persistent forth appears, 2ml of extract with few drops of olive oil and shaken vigorously then observed for the formation of emulsion
3.10.6 Detection of tannins
A small quantity of extract was mixed with water and heated on a water bath. The mixture was filtered and ferric chloride was added to the filtrate. A dark green colour was formed. It indicates that the presence of tannins.
3.10.7 Detection of carbohydrates
The plant extracts 0.5 mg was dissolved individually in five ml distilled water and filtered. The filtrates were used to test the presence of carbohydrates.
3.10.8 Detection of protein
To 0.5 mg of extract equal volume of 40% NaoH solution and two drops of one percent copper sulphate solution was added. The appearance of violet colour indicates that the presence of protein.
3.11Quantification of bioactive compounds
3.11.1 Flavonoid test (van – Burden and Robinson, 1981)
1.0ml cultural filtrates was mixed with 4ml of distilled water and subsequently with 0.30ml of a NaNO2 solution (10%). After 5 min, 0.30ml AlCl3 solution (10%) was added fallowed by 2.0ml of NaOH solution (1%) to the mixture. Immediately, the mixture was thoroughly mixed and absorbance was then determined at 510 nm versus the blank. Standard curve of quercetin was prepared (0-12mg/ml) and the results were expressed as quercetin equivalents (mg quercetin/gm dried extract).
3.11.2 Saponins (Nahapetian and Bassiri, 1975)
Two ml of cultural filtrates were mixed in 100ml of 20% ethanol. This sample suspension was heated over water bath for 4 hour at 55ºC with continuous stirring. This sample was filtered and extract was collected in 200ml capacity of beaker. Obtained residue re- extracted with 100ml of 20% ethanol. Combine extracts heated over water bath at about 90 till volume was reduced to 40ml. The concentrate was transferred into a 250 ml separating funnel and 10 ml of diethyl ether was added and shaken vigorously. The aqueous layer was recovered while the ether layer was discarded. The purification process was repeated. 30 ml of n-butanol was added. The combine n- butanol extracts were washed twice with 10 ml of 5% aqueous sodium chloride. The remaining solution was heated in a water bath. After evaporation the sample were dried in the oven and weighted.
100 mg of sample was hydrolysed in a boiling tube with 5 ml of 2.5 N HCl in a boiling water bath for a period of 3 hours. It was cooled at room temperature and solid sodium carbonate was added until effervescence ceases. The contents were centrifuged and the supernatant was made to 100 ml by using distilled water. From this 0.2 ml of sample was pipetted out and made up the volume to one ml with distilled water. Then one ml of phenol reagent was added and followed by 5.0 ml of sulphuric acid. The tubes were kept at 25-30?C for 20 min. The absorbance was read at 490 nm (Krishnaveni et al., 1984).
3.11.4 Proteins (Lowry et al., 1951)
The cultural filtrates mixed with 50 ml of 50% methanol (1:5 w/v) at 25°C for 24 hrs and centrifuged at 7,000 rpm for 10 min. 0.2 ml of bark extract was pipette out and the volume was made to 1.0 ml with distilled water. 5.0 ml of alkaline copper reagent was added to all the tubes and allowed it to stand for 10 min. Then 0.5 ml of Folin’s Ciocalteaus reagent was added and incubated in dark for 30min. The intensity of the colour developed was read at 660 nm.
3.11.5 Steroids (Chanwitheesuk et al., 2005)
1ml of filtrates was transferred into 10 ml volumetric flasks. Sulphuric acid (4N, 2ml) and iron (III) chloride (0.5% w/v, 2 ml), were added, followed by potassium hexacyanoferrate (III) solution (0.5% w/v, 0.5 ml). The mixture was heated in a water-bath maintained at 70±20°C for 30minutes with occasional shaking and made up to the mark with distilled water. The absorbance was measured at 780 nm against the reagent blank. ?-Estradiol is used as a standard material and compared the assay with ?-Estradiol (concentration 20 µg) equivalents.
3.11.6 Terpenoids (Indumathi et al., 2014)
The one ml of filtrates was taken and soaked in 9ml of ethanol for 24 hour. After filtration the extract was extracted with 10mL of petroleum ether using separating funnel. The ether extract was separated in pre-weighed glass vials and waited for its complete drying (wf). Ether was evaporated and the yield (%) of total terpenoids contents was measured by using the following formula (wi-wf/wi×100).
3.12 Partial purification of enzyme
Yeast isolates was cultured for 24 – 48 hours in yeast malt broth at 37°C. After incubation, the culture centrifuged at 10,000 rpm for 15 min at 37°C. The supernatant separate from the pellet containing cell debris. The ammonium per sulfate will add to the culture supernatant to get 75 % saturation level from initial zero concentration. Then the precipitated solution was recentrifuged at 6000rpm for 10-15 min at 37°C. The pellet obtained after centrifugation dissolved in minimum volume of 50mM Phosphate buffer, pH 7.0. Overnight dialysis was performed using 10,000 MW cutoff dialysis bag for the precipitate against 0.001 phosphate buffer with respective three changes of same buffer, dialysis will centrifuged to 10,000 rpm at 37°C. The pellet was separated and freeze dried.
The lyophilized enzyme dissolve in minimum amount of 0.001 M phosphate buffer and applied on to the sepharosecolumn (1.5 X 25 cm) previously equilibrated with 20mM Phosphate buffer. The fractions collected by eluting the enzyme using by 50mM phosphate buffer. The active fractions collected by measuring the optical density at 280 nm. The small aliquots collected fraction was tested for enzyme activity by iodine micro plate method. Fractions showing amylase activity pooled and again applied to (1 X 20 cm) DEAE-Cellulose matrix column (Bangalore genei). The column previously equilibrated with 50mM phosphate buffer pH 7.0 and the enzyme bound to the column eluted with a linear gradient of 2ml/ hrs fractions collected and checked for enzyme activity.
3.13 Characterization of enzyme production by potential yeasts (Buzzini and Martini, 2002)
3.13.1 Effect of temperature on enzyme activity
To determine temperature activity profile for ?-amylase enzyme, assay was carried out at different temperature i.e 20, 25, 30 and 35°C.
3.13.2 Effect of pH on enzyme activity
For determination of suitable pH range for enzyme activity on various pH i.e. 5, 5.5, 6.0 and 6.5 adjusted to 0.1M sodium hydroxide and hydrochloric acid was used.
3.13.3 Effect of Incubation Time on enzyme activity
For determination of suitable incubation time range for enzyme activity on various incubation time period viz., 0, 24, 48, 72, 96 and 120 hrs was used.
3.13.4 Effect of NaCl Concentration
Salinity Malt extract broth in triplicate was prepared using sea water of different salinities (0, 5, 6, 7 and 8 %).
3.13.5 Effect of Nutrient Source
For effective growth of identified strain on yeast malt extract media at optimized growth conditions, sucrose, beef extract, dipotassium hydrogen phosphate, magnesium sulphate, calcium chloride and L-Asparaginase in different concentration of 0.25, 0.50, 0.75 and 1.0 mg/l. Yeast strain are inoculated in yeast malt broth contain different nutrient source individually and incubated at 37°C for 48 hrs. Samples were withdrawn at regular time intervals and analyzed for absorbance at 600 nm.
3.14 Molecular characterization study
3.14.1 DNA isolation from yeast strains (Doyle, 1990)
DNA from yeast strains was isolated by modified CTAB method. Overnight grown yeast cultures in YEPD broth were centrifuged at maximum speed. About 10mg of yeast cells for each strain were taken and pre warmed 200 µl of solution I at 65°C containing 1.4M NaCl, 2% CTAB, 20mM EDTA (pH 8.0), 0.2% ?-mercaptoethanol and 100mM TrisHCl (pH 8.0) was introduced, mixed well and incubated at 65°C for 15-20 minutes in water bath. After incubation, all tubes were cooled for 3-5 minutes and same volume of solution II (Chloroform: Isoamyl alcohol, 24:1) was added, mixed thoroughly and centrifuged at 14,000 rpm for 10 minutes at room temperature. Aqueous phase (upper) were taken from each eppendorf separately and 3M Na acetate (1/10) was introduced in each eppendorf along with equal volume of cold iso-propanol or double volume of cold absolute ethanol, mixed it gently and placed on ice for 10 minutes. All tubes after incubation were centrifuged at 12000 rpm at 4°C for 15 minutes and supernatant was disposed off. Five hundred microlitre of chilled 70% ethanol (solution III) was added directly for washing pellet and then centrifuged at 14000 at 4°C for 2 minutes. The pellet was air dried after discarding supernatant from each tube. The pellet was resuspended in 50µl double deionized water or TE-buffer to store at ?20°C. The yield of DNA was quantified by Spectrophotometer (Sambrook et al., 2004).
3.14.2 Amplification of 18S-ITS region by polymerase chain reaction (White et al., 1990)
Amplification of 18S-ITS region of rRNA gene was done by using ITS1 (F) 5’TCCGTAGGTGAACCTGCGG3′ and ITS4 (R) 5’TCCGTAGGTGAACCTGCGG3′ primers in thermocycler (Bioerxp cycler). The reaction mixture contained 100ng DNA, 5µl of 10 pmol each oligonucleotide primer, 3µl of 25mM MgCl2, 3µl of 250mM dNTPs mixture and Taq DNA polymerase (5units) in a total volume of 50 µl. PCR conditions were as follow: 3 min. at 94°C followed by 35 cycles (45 sec. at 94oC, 45 sec. at 55°C (annealing temperature), 1 min. at 72°C and final extension for 7 min. at 72°C. The amplified product was checked by running on 0.8% agarose gel and visualized by using UV illuminator and photographed.
3.14.3 DNA sequencing
Out of total amplified PCR products were selected based on the restriction pattern of both restriction endonuclease enzymes and sent to Center for Advance Molecular Biology (CAMB) Lahore, Pakistan for sequencing by automated sequencer. Sequenced data obtained was blasted on NCBI and submitted to Bankit for accession numbers.
3.14.4 Phylogentic analysis (Swofford, 2002)
Phylogenetic analyses were conducted with parsimony, Bayesian and distance analyses with individual genes as well as concatenated datasets. Maximum parsimony analyses were performed with PAUP 4.0b10. Bootstrap values for the most parsimonious tree were obtained from 1000 replications. Bayesian Markov chain Monte Carlo (B-MCMC) analyses were performed with MrBayes v3.0b4 (Ronquist and Huelsenbeck 2003). The analysis consisted of 1 000 000 generations of four chains sampled every 10 generations; the first 100 000 generations were discarded as burn-in, and the remaining trees were used to obtain a majority rule consensus tree for estimating the posterior probability of the branches. Neighbor joining analyses were conducted using PAUP 4.0b10 with the Kimura 2 parameter option.
3.14.5 Statistical Analysis
The Shannon-wiener diversity, Peilou’s evenness, Species richness and Species dominance were analyzed using PRIMER V5 (Clarke & Gorley, 2001). Diversity index provides a good measure of the community composition along with its survival strategy. Pearson coefficient correlation was determined in physico-chemical properties vs total number of yeast isolates by using SPSS software.
4.1 Isolation of yeast from water and soil samples
Isolation of yeast was isolated from four different types of water and soil samples collected from Manakkattai, Saviyarmunai, Vauvalthottam and Deveraraul of Muthupettai District. The Manakkattai area samples isolated 32 colonies from water sample and 35 colonies isolated from soil sample. The minimum number of yeast isolated from Saviyarmunai area (18 in water and 22 in soil). The maximum number of yeast isolated from soil samples compared from water samples were recorded respectively (Table 1; Plate II and Fig. 2 & 3).
4.2 Physicochemical properties of water samples
Physico-chemical properties of water samples were analysed in Manakkattai, Saviyarmunai, Vauvalthottam and Deveraraul of Muthupettai District. In Manakkattai area, air temperature (24.8°C), water temperature (26.6°C), transparency (23.2 cm), dissolved oxygen (2.0 mg/l), pH (7.3), salinity (42.5 ppt), total alkalinity (110 mg/l as CaCO3), total hardness (106 mg/l as CaCO3), electrical conductivity (13.5 mS), total dissolved solids (12.3 ppt), ammonia (0.3 mg/l), nitrite (0.02 mg/l), nitrate (0.09 mg/l), orthophosphate (0.4 mg/l), iron (0.1 mg/l), sulphate (25.2 mg/l), manganese (0.1 mg/l) and zinc (28.3 mg/l) were recorded respectively. Saviyarmunai water sample analysed in air temperature, water temperature, transparency, dissolved oxygen, pH, salinity, total alkalinity, total hardness, electrical conductivity, total dissolved solids, ammonia, nitrite, nitrate, orthophosphate, iron, sulphate, manganese and zinc was 32.1°C, 31.4°C, 24.8 cm, 3.3 mg/l, 8.4, 33.0 ppt, 194 mg/l, 159 mg/l, 16.8 mS, 15.9 ppt, 0.8 mg/l, 0.1 mg/l, 0.4 mg/l, 0.7 mg/l, 0.5 mg/l, 23.7 mg/l, 0.2 mg/l and 26.0 mg/l tabulated correspondingly.
Similarly, Vauvalthottam was 30.8°C, 27.4°C, 24.6 cm, 2.9 mg/l, 8.2, 38.2 ppt, 151 mg/l, 125 mg/l, 10.4 mS, 15.1 ppt, 0.8 mg/l, 0.1 mg/l, 0.2 mg/l, 0.6 mg/l, 0.3 mg/l, 26.1 mg/l, 0.09 mg/l and 24.3 mg/l as air temperature, water temperature, transparency, dissolved oxygen, pH, salinity, total alkalinity, total hardness, electrical conductivity, total dissolved solids, ammonia, nitrite, nitrate, orthophosphate, iron, sulphate, manganese and zinc. Whereas, Deveraraul area, air temperature as 29.4°C, water temperature as 27.3°C, transparency as 22.4 cm, dissolved oxygen as 2.7 mg/l, pH as 7.9, salinity as 39.6 ppt, total alkalinity as 132 mg/l, total hardness as 119 mg/l, electrical conductivity as 12.9 mS, total dissolved solids as 14.5 ppt, ammonia as 0.7 mg/l, nitrite as 0.08 mg/l, nitrate as 0.1 mg/l, orthophosphate as 0.5 mg/l, iron as 0.6 mg/l, sulphate as 25.6 mg/l, manganese as 0.07 mg/l and zinc as 22.5 mg/l was recorded respectively (Table 2; Fig. 4- 6).
4.3 Physico-chemical properties of soil samples
Analysis the physicochemical properties of Manakkattai area of pH (8.06), electrical conductivity (0.28 dsm-1), organic carbon (0.23 %), organic matter (0.46 %), available nitrogen (102.2 mg/kg), available phosphorus (4.52 mg/kg), available potassium (138 mg/kg), available zinc (1.08 ppm), available copper (0.95 ppm), available iron (4.36 ppm), available manganese (2.24 ppm), cation exchange capacity (24.1 C. Mole Proton+/kg), calcium (13.2 C. Mole Proton+/kg), magnesium (7.5 C. Mole Proton+/kg), sodium (1.36 C. Mole Proton+/kg) and potassium (0.27 C. Mole Proton+/kg) were analyzed and recorded.
In, Saviyarmunai area, 8.21 of pH, 0.24 dsm-1 of electrical conductivity, 0.24 % of organic carbon, 0.48 % of organic matter, 104.5 mg/kg of available nitrogen, 4.16 mg/kg of available phosphorus, 145 mg/kg of available, potassium, 1.15 ppm of available zinc, 0.86 ppm of available copper, 4.56 ppm of available iron, 2.21 ppm of available manganese, 20.6 C. mole Proton+/kg of Cation Exchange capacity, the calcium, magnesium, sodium and potassium was 14.2, 7.3, 1.46 and 0.31 C. mole Proton+/kg were recorded respectively.
In pH, electrical conductivity, organic carbon, organic matter, available nitrogen, phosphorus, potassium, zinc, copper, iron, manganese, cation exchange capacity, calcium, magnesium, sodium and potassium was 8.16, 0.35 dsm-1, 0.21 %, 0.42 %, 107.5 mg/kg, 4.25 mg/kg, 156 mg/kg, 1.06 ppm, 0.68 ppm, 4.25 ppm, 2.85 ppm, 25.4 C. mole Proton+/kg, 14.0 C. mole Proton+/kg, 7.4 C. mole Proton+/kg, 1.34 C. mole Proton+/kg and 0.28 C. mole Proton+/kg were recorded correspondingly.
In Deveraraul area soil samples, pH, electrical conductivity, organic carbon, organic matter, available nitrogen, phosphorus, potassium, zinc, copper, iron, manganese, cation exchange capacity, calcium, magnesium, sodium and potassium (8.07, 0.31 dsm-1, 0.27 %, 0.45 %, 101.9 mg/kg, 4.26 mg/kg, 149 mg/kg, 1.54 mg/kg, 1.01 mg/kg, 4.61 mg/kg, 2.35 kg/mg, 23.4 C. mole Proton+/kg, 14.2 C. mole Proton+/kg, 7.1 C. mole Proton+/kg, 1.32 C. mole Proton+/kg and 0.31 C. mole Proton+/kg) were recorded correspondingly (Table 3; Fig. 7- 12).
4.4 Identification of yeast isolates
4.4.1 Morphological and microscopical characteristics of yeast isolates
Totally sixteen yeast culture were isolated from four different types of water and soil samples. The strain number is NJJUMS 1 to NJJUMS 16. Most of the yeast strains are white colour (NJJUMS 1, NJJUMS 3, NJJUMS 6, NJJUMS 11, NJJUMS 13, NJJUMS 14, NJJUMS 15 and NJJUMS 16), yellow colour (NJJUMS 9 and NJJUMS 10), red colour (NJJUMS 2 and NJJUMS 12), blue colour (NJJUMS 4), pink (NJJUMS 8) and white in cream colour (NJJUMS 5). Most of them strain ascospore absent especially NJJUMS 13 strain and true mycelium is present in NJJUMS 11 and 12 strains. The results were observed and tabulated (Table 4 and Plate III & IV).
4.4.2 Biochemical characteristics of different yeast isolated using carbon assimilating tests
Carbon assimilation test seven different types of carbon (glucose, maltose, sucrose, fructose, mannitol and starch) were performed. All yeast strains are utilized in glucose carbon source. Lactose source utilized in only four strains (NJJUMS 3, 4 and 14, 15), all yeast strain show positive results in maltose especially NJJUMS 1, 2, and 14. In the same way mannitol shows positive in all yeast strain, except NJJUMS 14, 15 and 16 strains. The results were observed and tabulated (Table 5).
4.5 Isolation of yeast from Muthupet seawater samples
The maximum percentage frequency was isolated from Candida elaebora, C.victoriae, C.sake, Candida sp., Rhodotorula glutinis and Saccharomyces cerevisiae (100% frequency). The minimum percentage frequency was also identified from Cryptococcus sp., Dioszella aurantiace and Endomycosis sp., (25 % frequency) recorded respectively in four different area samples (Table 6).
4.6 Isolation of yeast from from Muthupet soil samples
Totally sixteen yeast were identified in soil samples. The highest percentage yeast strain of Candida albicans, C.victoriae, C.sake, Rhodotorula glutinis, Saccharomyces cerevisiae and S. rosei (100 % frequency) recorded in all four stations and lowest percentage frequency were recorded in Dioszella aurantiace, Saccharomyces capensis and Saccharomyces sp. the results were calculated and tabulated (Table 7).
4.7 Pearson coefficient correction of physico-chemcial parameters vs population density
The physico-chemical properties vs population density was correlated in statistically. The positive correlation detected at 0.05% level of significance in both water and soil samples (Table 8 and 9).
4.8 Diversity indices of yeast isolates
In this study, generalized mean, Shannon diversity, Simpson diversity and Pielous Evenness index were calculated. The Shannon diversity was 0.034, Simpson diversity = 0.747 and Pielous Evenness index was 0.016 calculated in water samples. Whereas, the soil samples Shannon diversity was 0.038, Simpson diversity = 0.749 and Pielous Evenness index was 0.0661 recorded respectively. The above findings concluded that the study site had rich yeast diversity (Table 10 and 11).
4.9 Screening of enzyme activity by yeast strain
The primary screening of enzyme activity studies in eight yeast strains as Candida albicans, C.elaebora, C.sake, C.victoriae, Dioszella aurantiace, Rhodotorula glutinis, Saccharomyces cerevisiae and S.rosei was screened in five enzymes of amylase, protease, pectinase, esterase and cellulase. The amylase and protease enzymes studied in all yeast strain compared to other enzymes. The Saccharomyces cerevisiae and Rhodotorula glutinis screened in all enzymes, especially amylase, protease and cellulase enzymes are observed high zone formation as well as other yeast strain (Table 12 and Plate V).
4.10 Quantitative analysis of enzyme production of yeast
The quantitative assay was done in potential two yeast isolates (Saccharomyces cerevisiae and Rhodotorula glutinis). The enzyme activity recorded in S. cerevisiae of amylase (35.4 IU/ml), protease (31.5 IU/ml) and cellulase (32.5 IU/ml) and R.glutinis was 30.5 IU/ml of amylase, 29.2 IU/ml of protease and 31.8 IU/ml of cellulase enzyme production estimated (Table 13 and Fig. 13).
4.11 Qualitative analysis of bioactive compounds from potential yeast stain
The qualitative analysis of bioactive compounds saponin, tannin, flavonoids, terpenoid, carbohydrate, protein, steroid and alkaloid was present in Saccharomyces cerevisiae yeast. Even though saponin, tannin, carbohydrate, protein, flavonoid, terpenoid and steroid also present in Rhodotorula glutinis strain were analyzed (Table 14 and Plate VI).
4.12 Estimation of bioactive compounds from potential yeast strain
Saccharomyces cerevisiae was studied in saponin (4.5 µg/ml), flavonoids (7.3 µg/ml), terpenoids (3.6 µg/ml), carbohydrate (3.5 µg/ml), protein (4.3 µg/ml) and steroid (1.2 µg/ml) recorded respectively. While, R.glutinis saponin (7.4 µg/ml), flavonoids (9.9 µg/ml), terpenoids (4.3 µg/ml), carbohydrate (3.4 µg/ml), protein (2.1 µg/ml) and steroid (1.7 µg/ml) also recorded correspondingly (Table 15).
4.13 Partial purification of enzymes from S.cerevisiae
4.13.1 Amylase enzyme
Partial purification of amylase enzyme was carried out by crude extract followed by dialysis and ammonium sulphate precipitation method. The specific activity of the crude sample was found to be 5.2 IU/mg of dialysis sample and ammonium sulphate precipitated sample was found to be 6.2 IU/mg and 3.4 IU/mg respectively. The recovery was 100% in crude enzymes, 58.4% in ammonium sulphate fractionation and 33.1% in dialysis method was recorded. Similarly, purification was 1.0, 2.4 and 2.6 fold was recorded respectively (Table 16).
4.13.2 Protease enzyme
The total activity of the crude sample was 53.6, specific activity (3.2 IU/mg), total protein (15.7 IU/ml), purification (1.0 fold) and recovery (100), whereas, the ammonium sulphate precipitation found to be 49.6 IU/mg of total activity, 4.2 IU/mg of specific activity, 28.3 IU/ml of protein, 1.4 fold in purification and 43.4 % recovery recorded respectively. The dialysis level total activity as 22.3 IU/ml, specific activity as 2.4, total protein as 11.3 IU/ml, purification 1.6 fold and 34.1 % recovery calculated (Table 17).
4.13.3 Cellulase enzyme
The specific activity of the crude sample was found to be 3.9 IU/mg of dialysis sample and ammonium sulphate precipitated sample was found to be 4.8 IU/mg and 2.2 IU/mg respectively. The recovery was 100 % in crude enzymes, 45.6 % in ammonium sulphate fractionation and 30.0 % in dialysis method was recorded. Other hand the purification was 1.0, 1.4 and 2.3 fold was recorded correspondingly (Table 18).
4.14 Partial purification of enzymes from R.glutinis
In amylase enzyme purification studies, the total activity of the crude sample of 79.6 IU/ml, specific activity (3.8 IU/mg), total protein (41.7 IU/ml), purification (1.0 fold) and recovery (100), while, the ammonium sulphate precipitation as 54.6 IU/ml of total activity, 5.5 IU/mg of specific activity, 33.1 IU/ml of protein, 2.2 fold in purification and 48.4 % recovery recorded respectively. The dialysis level total activity as 35.3 IU/ml, specific activity as 3.9, total protein as 19.9 IU/ml, purification 1.6 fold and 23.1 % recovery calculated (Table 19).
Protease enzyme purification of R.glutinis by three methods. The ammonium sulphate method was more suitable method for the purification studies. The maximum recovery of protease enzyme recorded in ammonium sulphate method (48.2 %) followed by dialysis (30.9 %). The total protein of 29.1 IU/ml in ammonium sulphate precipitation, 25.3 IU/ml of dialysis method and 34.5 IU/ml of crude enzyme extract was analyzed in tabulated (Table 20).
Partial purification of cellulase enzyme was carried out by crude extract followed by dialysis and ammonium sulphate precipitation method. The specific activity of the crude sample was found to be 1.8 IU/mg of dialysis sample and ammonium sulphate precipitated sample was found to be 3.5 IU/mg and 2.6 IU/mg respectively. The recovery was 100% in crude enzymes, 50.4% in ammonium sulphate fractionation and 29.8% in dialysis method was recorded. Similarly, purification was 1.0, 1.2 and 1.3 fold was recorded respectively (Table 21).
4.15 Characterization of crude enzymes from S.cerevsiae
4.15.1 Optimization of physico-chemical parameters
In S. cereivisae, the maximum production of enzyme in 30°C of temperature in 26.8 IU/ml (amylase), 24.6 IU/ml (protease) and 24.1 IU/ml of cellulase enzymes, pH 6.0, sodium chloride was 6 % and incubation period 96 hrs. Minimum production of enzyme in temperature (35°C), pH (5.0), sodium chloride (8 %) and incubation period (24 hrs) was 22.3, 13.4, 12.4 and 29.0 IU/ml of amylase enzyme, 20.5, 11.2, 10.1 and 22.7 IU/ml of protease enzyme and 20.6, 11.0, 9.5 and 17.2 IU/ml of cellulase recorded correspondingly (Table 22 and Plate VII).
In R.glutinis, the maximum production of enzyme in 30°C of temperature (27.2 IU/ml in amylase, 21.3 IU/ml in protease and 23.1 IU/ml in cellulase), pH is 6.0 of 18.8 IU/ml of amylase, 15.1 IU/ml of protease and 15.5 IU/ml of cellulase enzymes, sodium chloride concentration of 6 % of 14.5 IU/ml, 11.9 IU/ml, 10.6 IU/ml of amylase, protease and cellulase enzymes and incubation period 96 hrs was more suitable for the production of enzymes 30.4 IU/ml of amylase, 27.4 IU/ml of protease and 27.1 IU/ml of cellulase enzyme recorded respectively (Table 23).
4.15.2 Optimization of nutrient source
184.108.40.206 Saccharomyces cerevisiae
Different concentration (0.25, 0.50, 0.75 and 1.0 mg/l) of nutrient sources such as sucrose, beef extract, dipotassium hydrogen phosphate, magnesium sulphate, calcium chloride and L- Asparaginase. In maximum production of 0.50 mg/l of nutrient source of all enzymes and minimum production observed in 0.25 mg/l of nutrient sources. The sucrose sources 27.9 IU/ml in amylase, 25.4 IU/ml in protease and 24.6 IU/ml in cellulase enzymes. Beef extracts of 27.2, 24.2 and 22.3 IU/ml of amylase, protease and cellulase enzymes production (Table 24 and Plate VIII).
220.127.116.11 Rodotorula glutinis
The maximum enzyme production detected in 0.50 mg/l of sucrose, beef, dipotassium hydrogen phosphate, magnesium sulphate, calcium chloride and L-Asparaginase and minimum production observed in 1.0 mg/l nutrient source (Table 25).
4.16 Evolutionary relationships
The evolutionary history was inferred using the Neighbor-Joining method. As in many groups of fungi, the use of a morphological form concept has resulted in the circumscription of many genera and families that are not monophyletic. However by using phylogenetic analysis of a multigene dataset one clade is well supported and corresponds with Saccharomycetaceae.
For molecular characterization, two yeast strains from each class were selected and partially sequenced. The nucleotide sequences obtained were blasted by NCBI database. The blast query revealed that yeast strains NJJMUS 11 of Rhodotorula glutinis (Accession no. MH107821) and NJJMUS 12 Saccharomyces cerevisiae (Accession no. MH104611) deposited NCBI respectively (Fig. 14 & 15). In the evaluation relationship of the other species of Rhodotroula sp. was very close with the test yeast strain Rhodotroula glutinis (100 %) by BLASTN analysed, whereas S.cerevisiae (100 %) related with other species of Saccharomyces also very closely related by BLASTN analysed. The topology of the NJ tree inferred from the whole dataset (Fig. 16).
The genera Saccharomyces and Pichia have been found in both western and northeastern coastal waters of Taiwan (Tien and Wang, 2004). Only Candida, the most frequently recovered genus was isolated from seawater along the east coast. In the present study, Candida tropicalis was the most frequently isolated yeast species found in the coastal waters of northeastern Taiwan. Species such as C. tropicalis and Rhodotorula rubra have been found to occur widely in the marine environment. C.tropicalis has been found in the Indian Ocean waters and in the intestines of marine animals distributed in the Pacific and Atlantic Oceans (Kutty and Philip 2008, Wang et al. 2008).
The predominance of Cryptococcus species in soil, particularly arid soil, has been ascribed to their ability to produce polysaccharide capsules (Vishniac, 2006), but there may be additional reasons. Although yeasts, in general, utilize simple sugars, some basidiomycetous species may follow different assimilation patterns (Kurtzman and Fell, 1997). Arenz et al. (2006) found that Cryptococcus species accounted for 67% (4 out of 6) of the yeast species identified in the Dry Valley soil and 72% (13 of 18) in soils surrounding the historic huts. However, studies of yeasts from other locations did not show this predominance of basidiomycetous yeasts. For example, the yeasts isolated from Mid-Atlantic hydrothermal vents were dominated by ascomyceteous genera (12 of the 19 identified species) (Gadanho and Sampaio, 2005).
In the present study, total yeast was isolated from 103 CFU/ml in water samples and 117 CFU/ml in soil samples recorded respectively. In the finding are discussed in Meyers et al. (1967), the average population densities of oceanic, coastal and estuarine waters are reported to be less than 100 cells/l. Several thousand yeast cells per litre of water were observed in coastal waters. Majority of yeasts were obligate aerobes that require oxygen for growth and reproduction and hence yeasts do not inhabit anaerobic waters and sediments. But Kriss (1963) reported that yeasts could be observed in the anaerobic zone of the black sea.
Several decades ago, marine-occurring yeasts were isolated from estuarine and coastal sediments in western Taiwan (Cheng and Lin, 1977). The genera of yeasts classified included Saccharomyces, Torulopsis, Debaryomyces, Endomycopsis, Pichia, Kloeckera, and Rhodotorula. However, many rivers converge into the continental shelf sea of the Taiwan Strait between the densely populated areas of western Taiwan and northeastern China. Therefore, microbiological contamination may have occurred at the near-shore areas of western Taiwan, rather than from the Pacific Ocean off eastern Taiwan.
Candida exhibited a ubiquitous distribution, as it is the dominant genera in both Arabian Sea and Bay of Bengal and they are found in 200 m, 500 m and 1000 ml depths. Candida is one of the most common genera in marine environments (Fell, 1967). At the same time, Fell et al. (1963) also reported that Candida is found more often in coastal waters often in close proximity to urbanized regions where waters are highly polluted by domestic water.
Fell (1967) reported that the Somali Current had a significant effect on yeast abundance (Liu et al. 2000). A novel yeast species, Kazachstania jiainicus, was isolated from forest soil in Jiain, Hualein, which is far from sampling stations along the eastern coast of Taiwan. Saccharomyces yakushimaensis is another interesting case; it was initially isolated from soil on Yaku Island off southern Kyushu in Japan (Mikata et al. 2001).
Based on the colony characteristics (white and creamy texture) ovoid microscope shape, the presence of ascospore, and budding pattern (multipolar), the selected isolate (Date-juice and grapes) were found to belong sacharomyces type unicellular ascomycete according to Lodder (1971) and Boekhout and Kurtzman (1996).
In the current study, the morphological and microscopical study the isolated yeast are white, cream, rough, smooth, wrinkled, entire, undulating, slightly convex, round, oval shaped colour, surface, margin, elevation are seen and ascospore formation, pseudomycelium formation and true mycelium are present or absent observed respectively in both samples.
The isolates were tested for fermentation of carbohydrates and Date-juice strain was capable to ferment six sugars out of the seven sugars tested. Glucose, Sucrose, Fructose, Lactose, Maltose and Trehalose were successfully fermented by this strain but it can’t ferment Xylose. The Grapes failed to ferment Maltose and Xylose, but utilized five other carbohydrates, which proved the identity both of the microorganisms are Saccharomyces cerevisiae.
The diversity of species in each lake showed significant differences according to the Shannon index (p < 0.05). This index reveals that both lakes have low diversities because they are between 0 and 3, but that they are not equal because of the higher number of species found in the Central Lake (18 species). Possibly, the significant differences found in the physicochemical parameters between these two environments allow each lake to provide the resources and conditions necessary to sustain the same yeast species richness but not the same diversity of species (Bedoya et al., 2014).
In our study, diversity of yeast isolates were calculated for margalef’s index, mehninick’s index, pielou’s index, mcintosh’s evenness index, shannon’s index, margalef’s diversity and simpson’s dominance index and simpson’s diversity for both water as well as soil samples. The simposn’s diversity of water samples is 0.747 and 0.749 recorded respectively. This fact agrees with findings Magurran (1988) in which biodiversity often show a higher correlation with richness than with evenness.
On the contrary, Persiani et al. (1998) found that the biodiversity correlates better with evenness, because they had a variable number of species but evenness tended to be quite high in the majority of cases. A large number of species in a community is usually attributed to extensive niche differentiation (Frankland, 1981). In soil microfungal communities, an additional factor may be the historical diversity and capacity for survival (Christensen, 1981).
Hence, in present study to characterized in carbohydrate fermentation of sucrose, lactose, glucose, maltose, fructose, mannitol and starch observed in yeast isolates in both samples. All yeasts are capable to ferment in glucose and followed in sucrose, mannitol, maltose and lactose. But some yeast is fermented in starch substrate.
The yeast isolate BE showed the following distinguished characteristics such as globose cell shape, formed pseudohyphae, consisted of cylindrical cells shape, and branched chains. All isolates fermented glucose, galactose, sucrose, maltose, and trehalose. They assimilated sucrose, L-sorbose (slow), cellobiose (weak), melezitose, inulin (weak), ribitol, methyl-D-glucoside, citrate (latent), grew on vitamin-free medium, 10% NaCl with 5% glucose and grew on YM medium at 37°C. Therefore, they were identified as C. tropicalis (Modi et al., 2018).
The biochemical stated results, it was assumed that all the six isolates may be of yeast. Earlier, Sefa-Dedeh et al., (2003) and Chiang et al., (2006) also isolated Saccharomyces cerevisiae, Candida krusei, C. pelliculosa, C. glabrata, C. utilis, C. sphaerica, C. magnoliae, Rhodotorula mucilaginosa, R. glutinis and Cryptococcus laurentii yeasts from Tapai, a fermented rice beverage in Malaysia.
The marine soil is measured like the mainly significant issue in the marine atmosphere that influences the growth, replica and metabolic actions of biotic components including microbes. In the current study, the physico-chemical factor including pH, Electrical conductivity, Macronutrients like (organic carbon, nitrogen, phosphorus, potassium), Micronutrients like (iron, manganese, zinc, copper) and others Caution exchange capacity, Magnesium and Sodium of the soil samples were studied.
It is clear that the threat of ocean acidification on marine ecosystems and species represents a priority for future investigations and large-scale investments in green-energy sources. The most important challenge of research is to identify the vulnerability of some physiological processes of key marine species but also rate of tolerance and adaptation capability to the global climate change (Vaijayanthi and Vijayakumar, 2014 and Guinotte and Fabry, 2008).
The nature of soil in study area was Sand Clay Loam from all samples. In the present study, pH ranges from 6.3 to 6.9 the soil pH is slightly acidic in all soils. The pH of soil is one of the most important parameter. At basic or low acidic pH soils usually cultivated rice (Chandra Sharma, 2015).
The soil parameters like texture, calcium carbonate content, electrical conductivity (EC), pH, organic carbon (OC), available nitrogen, available phosphorus, available potassium iron, manganese, zinc, copper and cation exchange capacity were studied by the standard methods. Further, the correlation coefficient analysis between the physical and chemical boundaries of the marine soil samples and total actinobacterial isolates were performed (Manikandan and Vijayakumar 2015).
In the present study, the pH ranges from 7.3 to 7.9, salinity 33.0 to 42.5 ppt and water temperature in 26.6 to 31.4°C in water sample. Similar observations were reported by Padmavathi and Sathyanarayanan (1999) and Govindasamy et al. (2000) with direct relationship between salinity and pH, as shown by Bhave and Borse (2001). The high pH recorded during summer season could be due to the increase in temperature coupled with high salinity (Backnemo, 1981). These finding correlate with that the results of Nagarajaiah and Gupta, 1983 and Tiwari, 1990.
Analyses of seawater samples revealed salt concentrations from 2.5 to 3.6% and pH values from 8.1 to 8.3 (Chen et al., 2009). The high salinity and moderately alkaline conditions indicated that the collected waters rarely mixed with riverine water. Relatively few studies have investigated marine yeasts, and this group of Mycota is still poorly understood (Kutty and Philip, 2008). In total, 6 cultures were isolated from the seawater Samples. In four different types of genera, Saccharomycese, Saccharomycetes and Endomycosis sp. were identified.
The Nitrate Concentration ranged from 0.09 to 0.4 mg/l, nitrite in 0.02 to 0.1 mg/l, ammonia (0.3 to 0.8 mg/l), orthophosphate (0.4 to 0.7 mg/l), iron (0.1 to 0.6), sulphate (23.7 to 25.6 mg/l) and zinc in 22.5 to 28.3 mg/l in our study recorded. These finding agree with the observations made by Qasim (1980), Murugan and Ayyakkannu (1993) and Jagadeesan (1986) in Cochin Backwater and Uppanar Back waters of estury.
Soil characteristics such as pH 7.22-7.69, electrical conductivity 0.41 dSm-1 to 0.54 dSm-1, cation exchange capacity 16.3-23.8 C. mole proton+/kg, organic carbon 0.36-0.60%, nitrogen 2.74-3.29 mg/kg, phosphorus 1.1-1.28 mg/kg, potassium 4.13-5.34 mg/kg, zinc 0.53- 0.89 ppm, copper 0.53-1.97 ppm, iron 2.46-8.54 ppm, manganese 3.10-3.69 ppm, calcium 10.5-12.8 C. mole Proton+/kg, magnesium 6.7-9.2 C. mole Proton+/kg, sodium 1.33-2.98 C. mole Proton+/kg and potassium 0.14-0.28 C. mole Proton+/kg, showed variation during different seasons (Karthikeyan et al., 2013).
In the current study, physico-chemcial properties ranges in between pH (8.06 – 8.21), electrical conductivity (0.24 – 0.35 dsm-1), organic carbon (0.21 – 0.27%), organic matter (0.42 – 0.48%), available nitrogen (101.9 – 104.5 mg/kg), available phosphorus (4.16 – 4.52 mg/kg), available potassium (138-156 mg/kg), available zinc (1.06 -1.54 ppm), available copper (0.68 – 1.01 ppm), available iron (4.25 – 4.56 ppm), available manganese (2.21 – 2.85 ppm), cation exchange capacity (20.6 – 25.4 C.mole proton+/kg), calcium (13.5 – 14.2 C.mole proton+/kg), magnesium (7.1 – 7.5 C.mole proton+/kg), sodium (1.32 – 1.46 C.mole proton+/kg) and potassium (0.27 -0.31 C.mole proton+/kg) recorded respectively in all four sites and correlated in 0.01% level of significance.
Madhava Sarma (2015) studied crops vary to the degree of sensitivity to salts, but most crops tolerate levels of 1.1 or less with no effect on yield. Excess salinity may cause moisture stress within the plant. However, too pure of can also be detrimental. Water with too few salts can lead to surface soil dispersion and soil crusting. Salinity is a measure of the total amount of soluble salts in soil. As soluble salt levels increase, it becomes more difficult for plants to extract water from soil.
Yeast strains were classified as very good producers of pectin depolymerizing enzymes when presented clear halos around colonies of at least 15 mm (Kluyveromyces marxianus NRRL-Y-1109), good producers when the halos were of at least 10 mm (Pichia pastoris, Candida rugosa, Saccharomyces cerevisiae NRRL-Y-12632 and Kluyveromyces marxianus TEM-4), weak producers when halos were at least 5 mm (4 strains), poor producers when no pectinolytic activity and no clear lysis zones were observed (11 strains) (Oskay and Husniye, 2015). The yeast K. marxianus NRRL-Y-1109 was found excrete high level of extracellular pectinolytic activity on pectin agar and was selected for further studies.
Schwan et al. (1997) isolated 12 yeast strains from cocoa fermentations, among them only four showed extracellular pectinase activity. They also reported K. marxianus was the most pectinolytic activity.
In the present study the extracellular enzymes (amylase, protease, pectinase, esterase and cellulase) were screened in yeast strain. The maximum enzyme activity was observed in S.cerevisiae and Rhodotorula glutinis in above mentioned enzymes are revealed positive results. This finding is different with previously reported finding (Oliveira et al., 2007). Even though same nitrogen source was used, Oliveira et al. (2007) found that extracellular protein produced by the same rhizobia can differ significantly. However, this result is very influenced by intrinsic properties of the microorganism. For S. fibuligera, it might be that total extracellular protein production is highly affected by the nitrogen source in the media. This hypothesis needs to be confirmed by more experimental data.
The production of yeast proteases has so far been studied mainly for their implications in the beer and wine industry (Bilinski and Stewart 1990; Dizy and Bisson 2000; Strauss et al., 2001). Only a few yeast proteases have been studied for alternative potential applications (Ray et al., 1992). Pectin enzymes for industrial uses have so far been produced by moulds and bacteria (Sakai et al., 1993), the pectolytic activity of yeasts has, however, been studied with contrasting results (Charoenchai et al., 1997; Strauss et al., 2001).
Moreira et al. (1999) found that the amylase production by Aspergillus tamarii was higher at pH 6 while Nahas and Waldemarin (2002) observed the maximum amylase production by Aspergillus ochraceus at initial pH 5.0. Also, the result is in agreement with the result of Hostinova (2002).
Crude amylases produced by terrestrial yeast Saccharomycopsis fibuligera are capable of converting raw starch and cassava into trehalose effectively. Chi et al. (2009) reported that gluco-amylase produced by the marine yeasts was different from that produced by other yeasts and fungi. It may have great potential for use in direct digestion of raw potato starch in food and fermentation industries The results also suggest that marine yeast offer great potential for the production of novel enzymes which would not be observed from terrestrial yeasts and fungi (Gupta et al., 2003; Dariush et al., 2006).
In our study implicated that the quantification analysis of enzyme production of amylase, protease and cellulase was 30.5, 29.2 and 31.8 IU/ml of R.glutinis and 35.4, 31.5 and 32.5 IU/ml of S.cerevisiae measured.
Some researcher worked that the black yeasts are the major amylolytic, ureolytic, proteolytic enzyme producers. Even though many terrestrial yeasts belonging to the genus Candida, Yarrowia and Aureobasidium is reported to produce protease, only a single marine yeast strain Aureobasidium pullulans is able to produce protease (Ni et al., 2008). Proteases have many applications in detergents, leather processing, silver recovery, medical purpose, feeds, chemical industry as well as waste treatment (Kumar and Tagaki, 1999, Anwar and Saleemuddin, 1998).
L-Asparagine is a good source of carbon and nitrogen for microorganisms. It has long been believed that L-asparagine is deamidated only intracellular by L-asparaginase. In other words, the enzyme never appears outside the cell, although the reason for such specific localization inside the cell has been left unexplained.
Similarly, the production of L-asparaginase initiated after 24 hrs of incubation, maximum cell growth and enzyme activity was found after 6th day of incubation. A positive correlation was observed between cell growth and enzyme activity. The maximum L-asparaginase production was recorded for Modified glycerol asparagine broth (5.23 IU mL-1) followed by Modified ISP-2 broth (4.75 IU mL-1) and Asparagine dextrose salts broth (4.21IU mL-1). Amena et al. (2010) noted that maximum production of enzyme was attained by the strain Streptomyces gulbargensis at 120 hrs of incubation (Ushakiranmayi et al., 2014). In Streptomyces griseus NIOT-VKMA 29 maximum yield of L-asparaginase was obtained from 6th day old culture (Meena et al., 2015).
Ambi rani et al. (2012) reported the production of L-asparaginase in submerged fermentation wherein the maximum of 3.8 U/ml at 96 hrs of incubation period was attained. Further increases in incubation period didn’t show any significant increase in enzyme production rather it was decreased. Thus optimum time of enzyme synthesis was 96 hrs after inoculation. This is in line with our findings in majority of the seven microbes tested through submerged culturing. The authors of this paper, when worked with Aspergillus terreus maximum enzyme activity was obtained when pomegranate skin powder was used as substrate in solid culturing at 120 hrs of cultivation (Nair et al., 2013) and okara hydrolysate fortified with L-asparagine gave 63.89 U/ml of activity at 120 hrs in submerged cultivation (Kumar et al., 2013). Sarquis et al. (2004) reported that the highest L-asparaginase activity of A. terreus in liquid medium was found at 48 hrs while in solid medium the optimal period for enzyme production was 96 h. Only in sea water isolate, we found increased accumulation of enzyme activity till 160 hours of culturing.
Crude culture supernatant of bacterial ?-amylase contained about 6.98 U of enzyme activity per ml. After ammonium sulphate precipitation and desalting by dialysis, the enzyme preparation contained 32.75 U of enzyme activity per ml. This achieved a 4.69 fold in purification. Crude culture supernatant of fungal amyloglucosidase exhibited 0.52 U of activity per ml while it showed an enzyme activity of 2.93U per ml after ammonium sulphate precipitation and desalting by dialysis, 5.60 fold purification was attained (Adeniran and Abiose, 2011).
In the current study, the partial purification of amylase, protease and cellulase enzyme from S.cerevisiae and R.glutinis. The maximum enzyme was recovered in amylase in both yeast strain compared to protease and cellulase enzymes.
The pectinase secreted by K. wickerhamii has optimum conditions were as follows: pH 5.0, Temperature, 32°C and an incubation period of 91 h (Moyo et al., 2003). Earlier, the optimal conditions for PGase activity (9.8 U/mL) reported from K. marxianus were pH, 4.6 and temperature, 31°C when beet juice was the used as a substrate (Serrat et al., 2011).
The wild type yeast Wickerhamomyces anomalus (Pichia anomala) was able to produce PGase in liquid medium containing glucose and citrus pectin (51 U/mL) (Martos and Zubreski, 2013). PGase from Aspergillus niger in SmF, was strongly decreased when glucose or sucrose (3%) was added to culture medium containing pectin (Solis-Pereira et al., 1993).
Gomes et al. (2005) reported a wide range of pH (5.0 to 9.0) over which glucoamylase produced from Aspergillus flavus, maintained stability. In the same work, glucoamylase from Thermomyces lanuginosus showed stability between pH 7.0 and 8.0. Fossi et al. (2005) also submits that a thermostable amylase from ascomycetes yeast strain exhibited stability within a wide range of pH 3.0 and 8.0 after incubation at 30°C for 30 min. Partial purification of the crude amylase was done by ammonium sulphate precipitation & dialysis similar techniques have been used earlier by Yandri et al. (2010).
The purified enzyme was characterized for the effects of temperatures, pH, activators and inhibitors, 37°C was found to be the optimum temperature, pH 7 as optimum pH, Ca/Mg/Zn as good enhancers, SDS and EDTA as inhibitors: earlier also purified enzymes have been characterized for the effect of temperature, pH, activators and inhibitors by Kubrak et al. (2010) and Srivastava (1987).
However, in their study, culture medium with peptone (1%) was found best nitrogen source for enzyme production. It has been reported that maximum an extracellular PGase activity of Mucor circinelloides, was obtained in 48 h at 30°C and pH 4 with pectin methyl ester (1% wt./vol.) as carbon source and a combination of casein hydrolysate (0.1% wt./vol) and yeast extract (0.1% wt./vol) as nitrogen source (Thakur et al., 2010).
Effect of pH, temperature and NaCI concentration on PGase activity of K. marxianus were 5.5, 40-45°C and 1%, respectively. The optimum pH and temperature are also consistent with values that have been found for K. marxianus pectinase production of 5 and 40°C respectively (Schwan et al., 1997).
Based on the results of these experiments, the yeast strain K. marxianus produced PGase possessing different activity of pH, temperature, NaCI concentration and thermal stability. The enzyme solution of yeast in this study has specific properties that may be applied directly to fruit juice industries without the pH modification. Nevertheless, because of the temperature stability of the enzyme offer advantages at processing temperature of 40 – 50°C, this is sufficient for industrial process, although enzyme preparation could be improved (Oskay and Hüsniye, 2015).
The optimal pH range for growth of yeast can vary from pH 4.0 to 6.0, depending on temperature, the presence of oxygen and the strain of yeast. This likely is due to the optimum pH value for the activity of plasma membrane-bound proteins, including enzymes and transport proteins (Narendranath and Power, 2005). In our study the strain Date-juice can grow in a wide pH range from 2 to 10, but ph 5.0 showed to be the optimum pH for it.
The results proved both strains have resistance against higher osmotic pressure. Both strains showed their highest growth in 6% NaCl containing media and gradually lowest in 15% and 20% NaCl containing media.
Other media however, showed lower yields of enzymes which could be attributed to solution of complex ions which might have induced inhibitory effect on enzyme production. Various nitrogen sources, minerals and phosphates like KH2PO4, K2HPO4 has been found best for pectinase production (Vyas et al., 2005; Narayana et al., 2014; Anuradha et al., 2010).
This implies that the pH of the medium influences the growth of microorganisms and hence the enzyme production. Each microorganism possesses a specific pH range for its growth and activity. Maximum pectinase production 35.84 U/ml from Bacillus sp. has been reported at pH 5.0 by Laha et al., (2014). Highest pectinase activity (1.6 IU/ml) was observed at pH 6.0 from Bacillus sp. isolated from vegetable waste soil (Kaur et al., 2016).
Maximum pectinase titres were produced at 30°C i.e. (5.59 IU) whereas; least pectinase production was found at 45°C (2.31 IU) from Stenotrophomonas maltophilia P9. Incubation temperature is the most important physical factor which affects enzyme production dramatically and their stability. Maximum enzyme activity at optimum temperature may be due to the faster metabolic activity and increase in protein content and extracellular enzyme production in culture supernatant. At very low temperatures, membranes solidify and high temperatures damage microorganisms by denaturing enzymes, transport carriers and other proteins thus lowering enzyme activity (Willey et al., 2008).
Similarly, in our present study, the optimization of physico-chemical parameters of R.glutinis and S.cerevisiae yeast strains. The maximum production of enzyme activity measured in 30°C temperature, 6.0 pH, 96 hrs of incubation, 0.75 mg concentration of sucrose, beef, dipotassium hydrogen phosphate, magnesium sulphate, calcium chloride and L-asparaginase parameters.
Afterwards incubation beyond 72 hrs resulted in a decreased enzyme activity that could be due to depletion of nutrients available causing a stressed microbial physiology eventually resulting in an inactivation of enzyme (Flores et al., 1997). Maximum yield of pectinase (40 U/ml) was obtained after 72 h incubation from Bacillus sp. isolated from soil by Nithisha et al., (2016). Venkata and Diwakar (2013) found that 48 hours was the optimum duration for maximum pectinase enzyme activity (166U/ml) from Bacillus circulans isolated from dump yards of vegetable wastes.
A series of experiments had been conducted at different glucose concentration to determine the optimum condition for twenty-four hours alcoholic fermentation. In shaking condition, the Date-juice strain showed highest 5.93% production in 6.5% and 7% glucose level. In the same parameters the Grapes strain produced 5.93% in 6% glucose concentration. In the non-shaking condition, the Date-juice strain showed 5.53% production in 4% glucose level and the Grapes resulted maximum 4.1% in the same glucose concentration.
Use of concentrated sugar substrate is one of the ways to obtain high ethanol yield during fermentation. However, due to osmotic stress high substrate concentration is inhibitory to fermentation (Jones, 1981). But the production of high concentration of ethanol is frequently limited by the inhibitory effect on productivity of the fermenting microorganism exerted by the substrate, the concentration of which affects osmotic pressure (Van Uden, 1989).
Strains were identified to the species level by amplification of the 5.8S rDNA gene and flanking Internal Transcribed Spacer (ITS). The primer ITS1 and ITS4 were used for amplification conserved regions of 5.8S rDNA, resulted in product of >1.5kb fragments confirming that the isolate was yeast (Mishra et al., 2018). Similarly, Esteve-Zarzoso et al., (1999) and Lentz et al., (2014) also used 5.8S rDNA for the strains identification up to the species level. The tentative phenotypic identification of all six isolates was confirmed by genotypic characterization in which 5.8S rDNA sequence analysis of these isolates as NGL3A, NGL4A, NGL1B and RNS4Cconfirmed the strains S. cerevisiae, whereas rest two isolates were identified as W. anomalus (RNL1A) and R. Mucilaginosa (RNS4C).
In the present study, the yeast strain of S.cerecisiae and R.glutinis was isolated from 18s rDNA gene and identified ITS region of specific yeast strain. The ITS region of particular strain deposited in Gene bank. The accession no. of S.cerevisiae MH104611 and R. glutinis MH107821. Similarly, The tree is based on a combined SSU rDNA and D1/ D2 LSU rDNA dataset. The D1/D2 LSU rDNA region has been sequenced for almost all known yeasts as an identification tool and also to estimate phylogenetic relationships in the Saccharomycetales (Kurtzman and Robnett, 1998)
The analysis of the D1/D2 region sequences proved a very efficient technique to identify yeast species. It allowed identifying to species level with 99% or more percentage of identity for all isolates tested, when compared with sequences stored in GenBank and subsequently with sequences of the type of strains of each of their species. Several isolates coincided with species that have not yet been officially described by the researchers who discovered them (Issatchenkia sp. and Bullera sp.) and we expect to contribute with some data to the taxonomic description of these species (Bedoya et al., 2014).
Molecular characterization of the selected pectinase producing strain P9 was done at genomic level by using 16S rRNA gene technique Genomic DNA. Further these amplified 16S rRNA sequences of the bacterial strains was blasted using online tool (mBLAST NCBI). The isolate P9 showed 96% homology with Stenotrophomonas maltophilia. The 16S rRNA gene sequences of the isolate has been deposited to National Centre for Biotechnology Information (NCBI) gene bank using Bankit program and has been registered in the databases vide accession number Stenotrophomonas maltophilia P9 |MF443881|. The taxonomical identification was done by the phylogenetic tree construction and the comparison of bacterial strain sequences with other homologous bacterial sequences (Poonam and Nivedita, 2018).
6. SUMMARY AND CONCLUSION
In the present investigation suggested that the entitled on ”EXTRACELLULAR ENZYME PROFILING OF MARINE DERIVED YEAST ISOLATED FROM MUTHUPET OF TAMILNADU” was determined.
In recent years the new potential of using yeast as biotechnological sources of industrially revenant enzymes has stimulated a renewed interest in the exploration of extracellular enzymatic activity in various industries.
Totally 117 colonies of yeast from Manakkattai, Saviyarmunai, Vauvalthottam and Deveraraul soil samples was 103 colonies per CFU/ml yeast isolated from Manakkattai, Saviyarmunai, Vauvalthottam and Deveraraul area of Muthupet mangrove environment .
The maximum total number of colonies (117 CFU/ml) recorded in soil samples when compared with water samples (103) colonies represented respectively.
The analysis of physico-chemical properties of water samples were analyzed. The maximum physicochemical parameters from Manakattai water sample recorded when compared to other places of Saviyarmunai, Vauvalthottam and Deveraraul respectively.
Maximum physico-chemical parameters were recorded from Vauvalthottam than followed by Saviyarmunai, Deveraraul and manakkatai soil samples of Muthupet areas.
Identification of yeast isolates by their morphological and microscopical characters of colour, surface ,margin elevation, ascospore and pseudomycelium and true mycelium was performed for the identification of yeast isolates and it was named as NJJUMS 1 to NJJUMS 13 than followed the carbon assimilating test were observed respectively.
Isolation of yeast was identified after the biochemical investigation. It was Aureobasidium mulluns, Candida elaebora, C.victoriae, C.tanuis, C.zeylanoides, C.sake, Candida sp., D.crocera, Endomycosis sp., Leucosporidilla muscorum, L.fragaria, Rhodotorula glutinis and Saccharomyces cerevisiae were conformed from the respective places.
The maximum percentage of frequency (100%) was candida elaebora, C.victoriae, C.sake, Candida sp., Rhodotorula glutinis and Saccharomyces cerevisiae recorded with respective places of muthupet area seawater sample.
Isolation of yeast such as Aureobasidium mulluns, Candida albicans, C.victoriae, C.zeylanoides, C.sake, Cryptococcus hansenii, Dioszellsa aurantiace, Rhodotorula larvngis, R.glutinis, Saccharomyces cerevisiae, Saccharomyces capensis, Saccharomyces sp. and Torulopsis sp. were recorded from four different places respectively.
Statistically significant results were analyzed by person correlation co efficient of water physiochemical parameters vs. population diversity were performed whereas soil samples also statistically significant results recorded with respective parameters.
Diversity indices such as richness, evenness and diversity with Saviyarmunai, Vauvalthottam Manakkattai and Deveraraul area water samples of Muthupet areas recorded with simpson’s index, Shannon’s diversity, pielou’s evenness and margalef’s index was performed.
Whereas soil samples were also the same parameters with respective statistic all analysis was significant results represented respectively.
Screening programmes for the selection of yeast strain able to produce bioactive molecular continue to be an important aspects of biotechnology.
On the basis of frequency of yeast population, only right strains are chosen for amylase, protease, pectinase, esterase and cellulase of extracellular enzymes qualitatively tested.
Analyze of quantitative enzymes production of yeast, two yeast strains such as S. cerevisiae and R. glutinis were selected. R. glutinis yeast has to be producing the amylase, protease and cellulase was 30.3, 29.3 and 31.8 IU/ml production of extracellular enzymes respectively.
Whereas Saccharomyces cerevisiae was 35.4, 31.5 and 32.5 IU/ml of amylase, protease and cellulase enzymes production respectively. Among the two strains S. cerevisiae was maximum product ion of above all three enzymes when compared with Rhodotorula glutinis yeast strains.
From the analysis bioactive compounds (qualitative) with the potential strains of yeast were determined. The Saccharomyces cerevisiae was saponin, tannin, protein, flavonoids, terpenoids, steroids and alkaloids represented whereas saponin, tannin, protein, flavonoids, terpenoids, steroids and alkaloids recorded respectively.
The maximum production of qualitative bioactive compounds from the potential S. cerevisiae was 4.5, 7.3, 3.6, 3.5, and 1.2 ug/ml with saponin, tannin, protein, flavonoids, terpenoids and steroids production recorded whereas R. glutinis potential strain was 7.4, 9.9, 4.3, 3.4, 2.1 and 1.7 ug/ml with saponin, flavonoids, terpenoids, carbohydrate, protein and steroids content represented respectively.
The partial purification of three enzymes such as amylase, protease and cellulase enzymes was purified in crude enzyme extract, ammonium sulphate precipitation and dialysis method.
The three enzyme of amylase, protease and pectinase enzyme purified from both S.cereivisiae and R.glutinis strain. Amylase was high amount of total activity, specific activity, total protein, purification and recovery percentage recorded from both yeast strain when compared to other enzymes.
According to optimization of physiochemical barrier for the production of extracellular enzymes amylase, protease and cellulase form potential strain of R. glutinis with physical barrier of temperature 30?C, pH 6, NaCl – 7% and incubation period 96 hrs was more suitable for higher production when compared with other parameters and SSF fermentation of invitro condition.
The maximum suitable parameter likes temperature 25?C, pH-6.5, NaCl-6%and incubation period was 96 hrs for the production of amylase, protease, and cellulase enzymes from S. cerevisae potential strains by invitro LSF fermentations.
Optimization studies of nutrient sources for extracellular enzymes production by R. glutinis were performed. The nutrient sources of 0.25, 0.50,0.75,1.0ug/l for amylase, protease and cellulase enzyme production.
The 0.50 mg/l of sucrose, beef extract and dipotassium, hydrogen phosphate, magnesium sulphate, calcium chloride and L.asparaginase was more suitable for the production of enzymes and other parameters adapted.
The potential strain of yeast S. cerevisiae with different concentration of sucrose, beef extract dipotassium hydrogen phosphate, magnesium sulphate, calcium chloride and L. asparaginase were altered with different concentration 0.25, 0.50, 0.75 and 1.0 ug/l for the production of extracellular enzymes amylase, protease, and cellulase production respectively.
However, our investigation clearly revealed the potential of yeast strains S. cerevisiae and R. glutinis were isolated from mangrove marine environment for production of extracellular enzymes and actually represent a specific sources of several enzymes potential exploitable for biotechnological purposes.
The molecular characteristics of S.cerevisiae and R.glutinis were evaluated by PCR amplification of 18S rRNA and ITS region. The amplified product was separated by agarose gel.
The Gene bank accession no. of S.cerevisiae MH104611 and R. glutinis MH107821 and to construct Phylogenetic relationship in both the yeast strain.
The present study concluded that yeast was predominant microflora of soil and water samples. The cell morphology of the yeast cells under microscope is ovoidal to elongate, single or in pairs and budding cells are also recognized. In the isolated strains were screened for the introduction of three enzymes namely, amylase, protease and cellulase. Saccharomycese cereivisea and Rhodotroula glutinis are more potential yeast in industrial Microbiology. The marine yeasts are comparatively few and that this group of marine mycota is still poorly understood. Different kinds of immobilized enzymes reactors and multiphase reaction systems have greatly influenced the processes that require catalysis by amylase. The marine derived Saccharomycese cerevisiae and Rhodotroula glutinis was highest fermentative activity among the many yeast isolates and species isolated and allied to the enzyme production from various biomasses. The enzyme from the marine yeast strains has so many unique properties and many potential applications in biotechnology.