SCREENING AND CHARACTERIZATION OF POTENTIAL ETHANOL OXIDIZING YEAST ISOLATES FROM SUGAR-RICH ORGANIC WASTE RESOURCES USING SCHIFF’S-BASE REACTION

, and cane molasses spill from the sugar industry, provide a favorable habitat for ethanol-oxidizing microorganisms with ADH activity. The total ethanol oxidizing microbial population of organic waste resources differs considerably from the overall microbial community. Yeast from the natural habitats has a great potential for developing novel enzymes for industrial uses. Under this backdrop, the present study was focused on screening and characterization of potential ethanol oxidizing yeast strains from sugar-rich organic waste resources. Totally seventeen ethanol oxidizing yeast strains belongs to Pichia sp. (9 strains), Candida sp. (2 strains) and Debaryomyces sp. (2 strains), Hanseniaspora sp , Lachancea sp , Torulaspora sp, and Magnusiomyces sp. have been identified. The ability of strains to oxidize ethanol into acetaldehyde was verified by the Schiff’s-base reaction through p-rosaniline assay. The selected five potential ethanol oxidizing strains were identified by 18S rRNA gene sequence as Pichia kluyveri FYES5, Pichia kudriavzevii FYES6, Pichia kudriavzevii VYES2, Candida mengyuniae CSYES1, and Magnusiomyces capitatus CSYES2 with the p-r index of 2.64±0.40, 3.79±0.54, 3.73±0.69, 2.28±0.24, and 2.87±0.58 respectively at 28°C. This is the first report to identify ethanol oxidizing yeast strains from sugar-rich organic waste resources and by this study found that the strain Candida mengyuniae has a better ability to oxidize ethanol.


INTRODUCTION
Alcohol dehydrogenase (ADH) is a crucial enzyme in fermentable microorganisms that aid in converting sugar to ethanol (Asokumar et al., 2018;Piriya et al., 2012).Alcohol dehydrogenase is an oxidoreductase enzyme present in all prokaryotes and eukaryotes that assists in ethanol fermentation and catabolism (Karaoğlan et al., 2020; Simpson-Lavy and Kupiec, 2019).One of the most effective biotransformation processes utilized in the industry is bioreduction, mediated by alcohol dehydrogenase/reductase (Hu et al., 2020).Ethanol is a kind of primary alcohol produced as a by-product of glycolysis under anaerobic conditions by the ADH enzyme, which also performs the reversible reaction of ethanol to acetaldehyde with a reduction of NAD + to NADH under aerobic conditions after glucose depletion (De Smidt et al., 2008).However, ethanol to acetaldehyde oxidation occurs both with and without oxygen; they are most likely reactive in aerobic conditions (Tagaino et al., 2019).Alcohol dehydrogenase is related to the oxidation of ethanol to acetaldehyde by a microbiological group that includes Streptococci, gram-positive aerobic bacteria, and yeast (Sunanda et al., 2019).Yeast is a unicellular eukaryote that can tolerate high and low pH, temperature, ionic strength, heavy metals, and other external factors.Yeast cells have the potential to survive with or without nutrient supplement, however, it requires sugar-rich substrate for their growth.These special tolerance characteristics of yeast were due to its nature of sporulation.Thus the function of yeast cells persists for a long duration of time when used as a biocatalyst.Candida albicans, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Pichia stipites, Lipomyces starkeyi, Hansenula polymorpha, and C. utilis were the few yeast species containing ADH enzyme (Karaoğlan et al., 2020;Suwannarangsee et al., 2010;Mazzoni et al., 1992;Verduyn et al., 1988;Heick and Barrette, 1970).Organic wastes are sugar-rich and low-cost substrates for ethanol production, as well as a desirable environment for ethanol-tolerant fermentable microorganisms (Arshad et al., 2019;Tiwari et al., 2013).External factors and microbes' development usually damage fruits, and its low pH and high sugar content encourage yeast, molds, and acid-tolerant bacteria growth.Microorganisms affect the quality of fruits through the enzymatic reaction and production of lactic acid, acetic acid, ethanol, and acetaldehyde (Erkmen and Bozoglu, 2016; Barth et al., 2009).Fresh vegetables that are less processed support the growth of yeast responsible for rotting.The spoilage due to contamination may be because of exposure to polluted environments during cultivation or poor hygienic conditions in processing (Nguyen-the and Carlin, 2009).The dairy industry releases a huge volume of wastewater and active sludge.The treated sludge was used as a fertilizer, secondary feedstock for phosphate fertilizer granules, soil amendments and also possesses high respiratory, and dehydrogenases activity (Shi et al., 2021;Ashekuzzaman et al., 2019;Frąc et al., 2017).Molasses rich in sugar spilled over soil decompose and release organic acids (acetic, propionic, butyric, and lactic) that neutralize the base and carbonates of the soil and stimulate microbial activity (Escolar, 1967).Fermentable microorganisms from sugar industry's cane molasses, such as Pichia veronae and Candida tropicalis can ferment sugar into ethanol (Hamouda et al., 2016).The catalytic activity of ADH enzyme has been less important for the past few decades in the industry because of its poor availability, limited substrate scope, need for co-factors for activity, and low performance, researchers have overcome the limitation and now its use is currently increasing in industries In this study, to isolate and identify the potential ethanol oxidizing strain with ADH activity from nature for industrial purposes, suitable sugar-rich cost-effective organic waste resources were chosen.The total microbial population habituated in organic waste and the specific microbial populations were able to oxidize ethanol were investigated.The overall microbial population and the total ethanol oxidizing microbial population found in the samples were hypothetically tested using Wilcoxon signed rank test.

Collection of organic waste resources
Spoiled fruit waste: Spoiled fruit waste samples, including Mandarin (Citrus reticulata), Banana (Musa acuminata), Sapota (Manilkara zapota), Grapes (Vitis vinifera), Apple (Malus domestica), Cantaloupe (Cucumis melo var.cantalupensis), Pomegranate (Punica granatum) available in the fruit shop at Chinnalapatti, Dindigul District, Tamil Nadu, India, were collected in an aseptic manner in a Plastic Ziplock pouch and brought to the laboratory for the further analysis.Fruit peel waste: Fruit peel waste of Pomegranate (Punica granatum), Cantaloupe (Cucumis melo var.cantalupensis), Mosambi (Citrus limetta) available in the fruit shop at Chinnalapatti, Dindigul District, Tamil Nadu, India, were collected in an aseptic manner in a Plastic Ziplock pouch and brought to the laboratory for further process.The obtained fruit peels were shattered into tiny shreds and weighed approximately 250g, then placed into each fermentation glass bottle in a triplet.Each of the bottles were sealed with a rubber cork hooked to a glass tube holding a rubber tube for CO2 release, and the mouth end of the bottle was entirely sealed to sustain an anaerobic environment (Vullo and Wachsman, 2005).To dissolve carbon dioxide, the bottle's rubber tube was placed in a tray of water.The bottles were entirely covered in aluminum foil to keep the environment dark, which promotes anaerobic ethanol production.The samples were fermented for approximately four months.The procedure was then carried out in aerobic conditions for a month to induce ethanol oxidizing microorganisms.Spoiled vegetable waste: A collective of spoiled vegetable waste samples such as Peas (Pisum sativum), Bulb onion (Allium cepa L.), Small onion (Allium parvum), Tomato (Solanum lycopersicum), Carrot (Daucus carota), Potato (Solanum tuberosum), Beetroot (Beta vulgaris), Radish (Raphanus sativus) available in the vegetable market at Chinnalapatti, Dindigul District, Tamil Nadu, India, were collected in an aseptic manner in a Plastic Ziplock pouch and brought to the laboratory for the further analysis.Raw sludge and composed sludge of dairy industry: Raw and composed sludge of dairy industry samples from SPS Dairy and Food Industry, Nagaiyargundampatti located at Dindigul District, Tamil Nadu, India were collected aseptically in an airtight Plastic Ziplock bag and brought to the laboratory for the further analysis.Cane molasses spill of sugar industry: The boundaries of the soil amended with cane molasses spill of the sugar industry near Alanganallur, Madurai were initially identified and collected aseptically in an airtight Plastic Ziplock bag and transported to the laboratory for further examination.The soil sampling were conducted following the selected systematic composite design (USEPA, 2002) as shown in figure 1.A set of n × g (6×4) individual samples were collected, where n is the number of composites and g is the number of individual samples used to form each composite.The set of 6×4 individual samples was systematically grouped within grid blocks into 6 composite samples.Each composite sample was thoroughly mixed and homogenized.From each composite sample, one or more subsamples were collected and further processed for enumeration of microbial population.

Enumeration of the total microbial population of sugar-rich organic waste resources
The Total Plate Count (TPC) technique was used to count the total microbial diversity in spoiled fruits, fermented fruit peels, and spoiled vegetables.An extract was obtained by vortexing each sample individually in a pre-sterilized 0.85% saline (Aneja, 2007), from which 10 mL of individual sample was aseptically suspended in 90 mL of saline (0.85%).Similarly, the Total Plate Count method was used to count the total microbial population in aseptically collected raw and composed sludge of the dairy industry as well as soil samples of cane molasses (Carter and Gregorich, 2007;Foght and Aislabie, 2005).The sample should not dry out and should be analyzed within 24 h of collection.To prevent heterogeneity, 10 g of the sample was aseptically transferred to 90 mL of 0.85% saline with a sterile spatula (Foght and Aislabie, 2005).All the suspensions were thoroughly mixed for 1 hour with the help of the orbital shaking incubator (REMI instrument Ltd.) before being cultured in three replicates after being serially diluted 10 -1 to 10 - 7 on respective medium (Tab 1) using the spread plate method.

Total ethanol oxidizing microbial population of sugar-rich organic waste resources
Total ethanol oxidizing microbial population viz., bacteria, yeast & molds, yeast, and actinomycetes of spoiled fruits, fermented fruit peels, spoiled vegetables, raw and composed sludge of dairy industry and soil amended with cane molasses were counted in triplicate on appropriate plates using the Total Plate Count technique (Carter and Gregorich, 2007; Foght and Aislabie, 2005) as described in table 1.The microbial colonies capable of oxidizing ethanol were counted using Schiff's reagent (Conway et al., 1987).Schiff's reagent was a combination of p-rosaniline and sodium bisulphite.Conversion of ethanol into acetaldehyde in the presence of ADH enzyme was examined by preparing media with the addition of 2 mL of prosaniline reagent (0.0025 g mL -1 p-rosaniline in ethanol (95% (v/v)) and 0.025 g of sodium bisulphite to 100 mL suitable agar media under 45 •C that suspended with 0.5% (v v -1 ) ethanol 0.1% (w v -1 ) carbon source.p-rosaniline reacting with NaHSO3 gets converted into the leuco form, making the media appear as a rosecolored medium.On agar plates, magenta-colored microbiological colonies indicate positive ethanol oxidation by ADH, whereas white-colored colonies suggest a negative outcome.If the organisms can oxidize ethanol to acetaldehyde, the leuco dye in the medium interacts with acetaldehyde to generate Schiff's base (red color).The reaction indicates that the equilibrium shifted ADH towards acetaldehyde synthesis that is dissolved and disseminated in an agar medium.The microbial count value of every sample measured thrice was reported as log cfu g - 1 .Significant differences between total microbial counts and ethanol oxidizing microbial count in sugar-rich organic waste resources were determined using Wilcoxon signed rank test.This test is used to compare the two related measurements of the same sample obtained under different conditions (Scheff, 2016).

Morphological, physiological, and biochemical characteristics of Yeast
For the morphologial identification the yeast colony color, texture, surface, elevation, and margin grown on Yeast-extract Peptone Dextrose (YPD) agar plate was noted.Under the microscope yeast cell shape, cell size (mean of 20 cells) measurement using ImageJ software, mode of reproduction, hyphae formation, and germ tube formation was observed and recorded according to Kurtzman et al. (2011).Carbohydrate fermentation, growth assimilation on carbon and nitrogen compounds, and other growth tests includes growth in vitamin-free medium and vitamin requirements, growth at high osmotic pressure, growth at 37°C and 45°C, acid production from glucose, starch formation, urea hydrolysis, 1% acetic acid tolerance were the physiological and biochemical characteristics examined for the isolated yeast cells following Kurtzman et al. (2011) methods.

Qualitative screening for ADH activity by Schiff`s based basal medium for yeast under aerobic condition
The conversion of ethanol to acetaldehyde on oxidation by ADH under aerobic conditions was examined by Schiff`s based basal medium for yeast (Conway et al., 1987;Wickerham, 1951).A loop of YPD slant culture of 24-48 hours was suspended in 2 mL of sterile distilled water in a sterile Eppendorf and evenly dispersed by the cyclic mixer (REMI equipments pvt Ltd.).The suspension was aseptically transferred into a basal medium (Wickerham, 1951) supplemented with 2% absolute ethanol to induce ADH expression in a serum bottle and incubated at 28°C in a shaking incubator (REMI Elektrotechnik Ltd.) with agitation 100 rpm under dark conditions for 3 days.Samples were prepared in triplicate and analyzed for ADH activity.Yeast cells grown in the basal medium were harvested by centrifugation process using a centrifuge (REMI Elektrotechnik Ltd.).The medium was taken for centrifugation process for 20 min at 3000 rpm (4°C).The collected cell debris was washed thrice and resuspended in sterile saline (0.85%).Further, the cell debris was dropped onto the aldehyde indicator plate containing Schiff`s based basal agar medium supplemented with 2% absolute ethanol and incubated at room temperature under dark condition.Development of magenta color in and around the colony indicates the conversion of ethanol to acetaldehyde which was examined at 20 minutes' interval from the time of inoculation till 12 hours and for 2 to 3 days.

Qualitative screening for ALDH activity by purple broth
The conversion of ethanol to acetic acid in presence of ALDH under aerobic conditions was examined by use of purple broth (Pavlova et al., 2013;Wickerham, 1951).To confirm whether the yeast isolates produce ADH along with ALDH, the cell suspension of about 100 µL was added to the sterile 10 mL purple broth supplemented with 2% absolute ethanol and placed under dark condition for 2-3 days at 28°C.The color change from purple to yellow indicates the conversion of ethanol to acetic acid.

Quantitative screening of ADH activity by p-rosaniline assay
Yeast cells grown for 2 to 3 days in a basal medium supplemented with 2% ethanol were harvested using a centrifuge (REMI Elektrotechnik Ltd.) at 3000 rpm at 4°C for 20 minutes.From the collected cell suspension, a loop of culture was smeared at the center of the aldehyde indicator plate and incubated at 28°C under dark conditions for 3 days.The colony spots of each strain grown on the aldehyde indicator plate containing Schiff's based basal agar medium supplemented with 2% absolute ethanol were recovered and transferred to 5 mL 0.85% saline solution.The suspended cells were homogenized vigorously by use of a cyclic mixer (REMI equipments pvt Ltd.) for 10 minutes.One aliquot was diluted into 1:100 dilutions in saline solution represented as 10 -2 dilution and used to determine the colony forming unit (cfu mL -1 ) by spread plate technique in basal agar medium supplemented with 2% absolute ethanol.Each homogenized cell suspension was made up to10 mL using absolute ethanol (99.9%)where p-rosaniline is miscible.The tubes were left over for 90 minutes at 28°C under dark conditions.After incubation, each cell suspension have been subjected to centrifugation for 5 min at 13, 000 rpm.The optical spectra were predicted by utilizing obtained supernatant.The optical spectra of leuco-rosaniline and shift base forms were determined at 420 nm and 548 nm.The appearance of high and less peak disappearance at the respective wavelength (λ) in magenta p-rosaniline form was observed using a spectrophotometer.A peaks at 420 nm and 548 nm were observed and the p-r index was calculated by the results of OD 548 nm/420 nm/ cfu mL -1 multiplied by the constant 10 5 .

Molecular sequencing of potential ethanol-oxidizing yeast isolates
The DNA of potential ethanol oxidizing strains were extracted and the purity was checked by running gel electrophoresis (1% agarose).The 18S rRNA genome was then taken from the samples and amplified by PCR using forward (NS1) and reverse primers (NS4).The amplified genome band comprised 1050 bp validated by gel electrophoresis.To eliminate impurities, the amplified sequence was filtered.On the ABI 3730xl Genetic Analyzer, forward and reverse DNA sequencing reactions of PCR amplicons were performed with NS1 and NS4 primers using the BDT v3.1 Cycle sequencing kit (Symbiont Life Sciences).The strains' forward and reverse sequences were combined via embossing merger.The resulting sequence (18S rRNA) was BLASTed against the NCBI nucleotide database.The sequences with a high percentage of similarity were categorized using the Maximum Likelihood technique and the Kimura 2-parameter model in MEGA X (Kumar et al., 2018, Kimura, 1980).

SEM analysis of potential ethanol oxidizing yeast isolates
The potential ethanol oxidizing yeast culture grown on YPD medium at 28°C for 2 to 3 days was centrifuged at 3000rpm for 20 minutes.The supernatant was discarded and added 4% of paraformaldehyde in 0.1M phosphate buffer (pH 7.4) to the cell debris and centrifuged again for 20 minutes at 3000rpm.Upon discarding the supernatant, the cell pellet was left without disturbing for 10 minutes after adding a small amount of 0.1M phosphate buffer (pH 7.4) (Al Shehadat et al., 2018).The step was repeated twice, and the suspension was re-centrifuged.The collected cell biomass was dehydrated with 30% alcohol, 50% alcohol, 70% alcohol, 80% alcohol, 90% alcohol, and 100% alcohol (twice).Time taken for each dehydration process was about 10 minutes.After dehydration, cell biomass was carried out for SEM (TESCAN) analysis.

Statistical analysis
The mean and standard error of the measured value was calculated by Microsoft Excel 2016.The normality test (Kolmogorov-Smirnov and Shapiro-Wilk test) and Wilcoxon signed rank test for the measured values were analyzed using the statistics program IBM SPSS statistics 23 (SPSS Inc., Chicago, Ill., U.S.A.).

Total ethanol oxidizing microbial population in sugar-rich organic waste resources
The investigation on ethanol oxidizing microorganisms was carried out under the idea of   The difference between the total microbial population and ethanol oxidizing population of similar groups statistically states that all those microbes that habited naturally on the chosen sugar-rich organic waste were not able to oxidize ethanol.The microbial population of spoiled fruits, fruit peel wastes, spoiled vegetables, and composed sludge shows a large difference in their ability to oxidize ethanol whereas raw sludge and cane molasses spill shows a medium difference in their activity.

Identification of ethanol oxidizing yeast from sugar-rich organic waste resources
Fruits naturally contain yeasts which perform a variety of functions depending on their environment and can also cause rotting (Gbeminiyi, 2022

Alcohol dehydrogenase and acetaldehyde dehydrogenase activity of ethanol oxidizing yeast strains
Acetaldehyde is a microbial bio-product, which is a metabolic catalyst of ethanol in ethanologenic microbes and it is very significant in the chemical industry (Kalnenieks et al., 2019).Microbial cells cultured in ethanol includes ADH enzyme, which may oxidize ethanol to acetaldehyde, as confirmed qualitatively by Schiff reagent (red color formation).Schiff reagent is a combination of p-rosaniline and sodium bisulphite (NaHSO3) that is commonly used to detect aldehydes, sugars on glycoproteins following periodic acid oxidation, and microbes that produce aldehydes in a broth test.The isolated ethanol oxidizing yeast strains were tested for ADH activity using Schiff's based basal agar medium in this investigation.ADH activity was regarded as positive in magenta-colored colonies with dye diffusion and negative in pink-to-white colonies with halo zones (Tab 7).Effectively oxidize primary and secondary alcohol rather (S)-N-benzyl-3-pyrrolidinol.According to the literature, this is the first investigation to be published that the strain P. kluyveri, P. kudriavzevii, C. mengyuniae, and M. capitatus has specific ethanol oxidizing activity, i.e, conversion of ethanol into acetaldehyde by enzyme alcohol dehydrogenase.

CONCLUSION
The current study shows that microbes adapted to sugar-rich organic waste resources can oxidize ethanol.The study statistically assured that not all the population of microbes habited in sugar-rich organic waste resources oxidize ethanol, only few of the isolates able to oxidize.Based on Schiff`s base reaction qualitatively the study explored that those species belonging to Pichia, Candida, Hanseniaspora, Lachancea, Torulaspora, and Magnusiomyces grow well in 2% ethanol as well as oxidize ethanol into acetaldehyde.The existence of ADH enzyme was confirmed in ethanol oxidizing yeast isolates by p-r index and potential strains were identified as Pichia kluyveri, Pichia kudriavzevii, Candida mengyuniae, and Magnusiomyces capitatus using 18S rRNA sequencing.These strains can be researched further for ADH-related applications such as pharmaceutical production, ethanol synthesis, aldehyde or ketone reduction, alcohol (primary and secondary) oxidation, and bio-sensing.In this study, it was demonstrated for the first time that Candida mengyuniae, a metsulfuron-methylresistant yeast strain, has unsuspected alcohol dehydrogenase activity that needs to be clarified more to allow subsequent research.
(de Miranda et al., 2022; Bartsch et al., 2020).Alcohol dehydrogenase was used as a biocatalyst in synthetic chemical production, for drug preparation in the pharma industry and ethanol-producing industry (de Miranda et al., 2022; Bartsch et al., 2020; Asokumar et al., 2018; Shinde et al., 2018).Cofactor-dependent alcohol dehydrogenase is less stable outside the cell and more expensive for biocatalysis.Thus, whole cells are frequently used as the biocatalyst (Hu et al., 2020) in various analytical instruments like biosensors.
a) A systematic composite design within a grid block (Adapted from USEPA, 2002).(b) Systematic grouping of each individual into composites and each composite into subsamples for analysis.
Legend: (+) -Positive; (-) -Negative; ND -Not detected Conway et al. (1987) were the first to develop an aldehyde indicator plate by incorporating Schiff reagent into a nontoxic solid medium.Para-rosaniline of Schiff reagent on reacting with NaHSO3 attains leuco form giving the medium a rose color.The medium's leuco form acts as a sink, forming Schiff's base and a strongly magenta color when it reacts with acetaldehyde, suggesting that the ADH enzyme changed the balance toward acetaldehyde generation (Tribelli et al. 2015).The aldehyde dehydrogenase activity's consumption of aldehydes and low levels of native alcohol dehydrogenase may contribute to the moderate color development in cell colonies on an aldehyde indicator plate (Conway et al., 1987).The adhB gene encodes the ADH II enzyme, Conway et al., (1987) cloned the adhB gene in Escherichia coli from Zymomonas mobilis.The adhB expression activity was detected directly by using an aldehyde indicator plate (Schiff's agar plate).Pavlova et al. (2013) tested acetaldehyde formation by Streptococcus gordonii adh mutants from ethanol directly on the PBB Schiff's agar plate.Tribelli et al. (2015) examined ethanol dehydrogenase activity in Pseudomonas extremaustralis on LB-Schiff`s based agar medium supplemented with ethanol and sodium octanoate.Rosca et al. (2016) screened yeast strains for acetaldehyde production by Schiff's reagent, which was validated by the creation of violet coloration.The present work screened selected ethanol oxidizing strains on Schiff's based basal agar medium supplemented with 2% ethanol.Pichia sp., Candida sp., Hanseniaspora sp., Lachancea sp., Torulaspora sp., and Magnusiomyces sp. can grow well in presence of 2% ethanol as well as oxidize ethanol into acetaldehyde effectively.The reaction time for ADH ranges from 20 min to 2 h at 37°C (Conway et al., 1987).However, the present testing reveals that three species of Pichia and one species of Candida shows ADH reaction within 20 min under the dark condition at 28°C whereas other yeast strain shows delay in their reaction time.Alcohol dehydrogenase (ADH) reduces ethanol to acetaldehyde and further to acetic acid ALDH.The full metabolization of ethanol into acetic acid occurs in microorganisms that have both active ADH and ALDH enzymes (Tribelli et al., 2015).The purple broth changes into yellow color as ethanol is converted into acetic acid in the medium, which lowers the pH.If microbes only contain the ADH enzyme and not the ALDH enzyme, they only create aldehyde without further conversion to acid from ethanol.Alam and Talukder (2015) used 5% ethanol to screen acetic acid-generating bacteria on yeast-extract peptone glycerol (YPG) culture supplemented with bromocresol purple as a pH indicator.Bacteria that can make acetic acid will have halos or a yellow tint around the colony.To assess the synthesis of ALDH and ADH enzymes, isolated ethanol oxidizing yeast were tested for acetic acid production in purple broth with 2% ethanol.Six species of Pichia, one species of Candida, and Hanseniaspora sp.contain ALDH activity, and the color changes from purple to yellow, but other yeast strains with ADH activity show a negative result and remain purple.Pichia sp. and Candida sp.produce acetic acid between 24 to 72 hours but Hanseniaspora sp produces it within 48 hours (Tab 7).Quantitative measurement of ADH activity of ethanol oxidizing yeastThe direct evaluation of ADH activity on an acetaldehyde indicator plate is a qualitative report.To pick out the effective ethanol oxidizing yeast strain among the isolated strains, adapted Tribelli et al. (2015) p-rosaniline assay method.To investigate any probability of oxidation reaction in LB infused with sodium octanoate extensively, Tribelli et al. (2015) performed a p-rosaniline experiment that revealed ADH activity.Magenta bacterial spot of Pseudomonas extremaustralis grown at 8 and 30°C in agar media plates treated with sodium octanoate showed a p-r of 5.97 ± 1.46 and 2.82 ± 1.33.In the present study, the magenta bacterial spot of FYES5 (Pichia sp.), and FYES6 (Pichia sp.) strains from spoiled fruits shows a p-r of 2.64±0.40 and 3.79±0.54,VYES2 (Pichia sp.) strain of spoiled vegetables and CSYES1 (Candida sp.), CSYES2 (Magnusiomyces sp.) from dairy industry grown on the aldehyde indicator plate, Schiff's based basal agar medium supplemented with 2% absolute ethanol shows a p-rosaniline index (p-r) of 3.73±0.69,2.28±0.24,and 2.87±0.58respectively at 28°C (Fig 2).

Figure 2
Figure 2 Absorption spectra of ethanol oxidizing yeast stains showing a minimal leco-rosaniline at 420nm and maximal shift base-rosaniline at 540nm in magenta rosaniline form and quantitative measurement of ADH activity by p-rosaniline assay: (a) Absorption spectrum and determination of p-rosaniline index of ethanol oxidizing yeast strains of spoiled fruits sample (b) Absorption spectrum and determination of the p-rosaniline index of ethanol oxidizing yeast strains of fermented fruit peel (c) Absorption spectrum and determination of the p-rosaniline index of ethanol oxidizing yeast stains of spoiled vegetable sample (d) Absorption spectrum and determination of the p-rosaniline index of ethanol oxidizing yeast strains of composed sludge sample from the dairy industry.

Figure 3
Figure 3 Phylogenic tree showing evolutionary relationship between the ethanol oxidizing yeast strains Pichia Kluyveri FYES5, Pichia Kudriavzevii FYES6, Pichia Kudriavzevii VYES2, Candida mengyuniae CSYES1, and Magnusiomyces capitatus CSYES2 based on 18S rRNA gene sequence.The species showing a high similarity percentage to the identified strains were retrieved from the BLASTn database and grouped by the Maximum Likelihood method and Kimura 2parameter model.Pichia kluyveri is a non-fermentable yeast that generates a lot of volatile chemicals.In wine production, P. kluyveri is one of the reasons for fragrance, glycerol, and ethanol synthesis, as well as inhibitory activity.Aromatic compound production by P. kluyveri is heavily regulated by culture medium, agitation, and temperature (Ciani et al., 2022; Yıldırım, 2021; Vicente et al., 2021; Méndez-Zamora et al., 2021).P. kudriavzevii is a yeast with teleomorphic characteristics.P. kudriavzevii, on the other hand, is a non-pathogenic and industrially significant yeast that is identical to Candida krusei, with infections varying depending on the environment from which the strain was isolated (Douglass et al., 2018; Cooper, 2011).P. kudriavzevii also acts as a plant growth promotor in the soil and forms biofilm (Ramya et al., 2021).Elahi and Rehman (2018) stated that P. kudriavzevii has both Pyruvate decarboxylase activity and alcohol dehydrogenase activity and can utilize hemicellulose and produces ethanol effectively.Chen et al. (2009) isolated Candida mengyuniae as a metsulfuron-methyl-resistant yeast strain from metsulfuron-methyl-contaminated soil.Alcoba-Florez et al. (2011) reported that C. mengyuniae causes intravenous catheter-associated fungemia.Magnusiomyces capitatus (Geotrichum capitatum) is a teleomorph yeast mainly isolated from woody areas, human & animal feces, and hot regions (Cooper, 2011).M. capitatus produces lipase and grow well in Olive mill wastewaters under controlled conditions (Salgado et al., 2020).M. capitatus derived from the trash of the palm oil industry produces extracellular and cell-bound lipase and is used as a biocatalyst in biodiesel production (Baloch et al., 2021).Microorganisms Geotrichum candidum, G. capitatum, G. eriense, G. fermentans, G. fragrans, G. klebahnii, and G. rectangulatum were able to produce ADH II with a high range of substrate specificity.This kind of enzyme can produce and reduce ketones and alcohol effectively.Fraser (1997) extracted (S)-1,3-butanediol dehydrogenase enzyme from G. candidum which has high stereoselectivity, oxidizing 1,3butanediol, phenylethanol, 3-hydroxybutyric acid ester and 2-octanol.Yamada-Onodera et al. (2007) purified ADH from G. capitatum which reduces N-benzyl-3-pyrrolidinone in presence of cofactor NAD + .Effectively oxidize primary and

Table 1
Dilutions, incubation temperature, and time preferred for enumeration of the respective group of the microbial population by Total Plate Count method.

Table 2
Total microbial population in sugar-rich organic waste resources NG -Not Detected; The values in the tables were the mean of three replicates with ± Standard Deviation (SD) Legend:

Table 3
Total ethanol oxidizing microbial population in sugar-rich organic wastes Sugar

-rich organic wastes Microbial load (log cfu g -1 ) Actinomycetes Yeast and molds Yeast Bacteria
ND -Not Detected;The values in the tables were the mean of three replicates with ± Standard Deviation (SD) Accordingly, in the present report, raw sludge from the dairy industry constitutes ethanol oxidizing actinomycetes and bacteria population of 4.61±0.02and 7.10±0.17log cfu g -1 whereas composed sludge comprising population load of actinomycetes, yeast & molds and bacteria of about 4.95±0.03,4.16±0.28and 7.28±0.06log cfu g -1 respectively (Tab 3).The total ethanol oxidizing actinomycetes population habited in soil amended with cane molasses lies around 3.42 ± 0.06 to 4.66 ± 0.02 log cfu/g (Tab 3).In the case of total yeast and molds, yeast and bacteria, the population from soil amended with cane molasses was not detected.The lack of microorganisms on Schiff's-based medium showed that those microorganisms habited were unable to survive in the presence of 0.5% ethanol and lack ethanol oxidizing capacity.Ethanol was non-toxic at low doses, but only a few microbial species survived at 0.1% ethanol concentration ( (Frąc et al., 2017) 2019)transparent liquid with a pronounced fruity fragrance that occurs naturally in the human body and plants.Acetaldehyde is also present in products like fruits and juice, vegetables, dairy products, bread, and beverages like tea and soft drinks, beer, wine, and spirits.Microorganisms such as yeast, and bacteria that consume these products have the potential to produce acetaldehyde, which acts as a direct precursor to ethanol, and alcohol dehydrogenase accumulates during respiration(Kalnenieks et al., 2019).Oxidation of ethanol to acetaldehyde by alcohol dehydrogenase is the main pathway of ethanol metabolism (Edenberg, 2007).The ethanol oxidizing actinomycetes, yeast & molds, yeast, and bacteria populated in spoiled fruit samples were found to be 4.84 ± 0.01, 5.37 ± 0.05, 5.56 ± 0.04, 7.36 ± 0.06 log cfu/g respectively by the Total Plate Count method (Tab 3).Fermented fruit peels pose ethanol oxidizing yeast & molds population lies around 4.26±0.24 to 5.31±0.03logcfug -1 and yeast about 5.20±0.17 to 6.34±0.02logcfug -1 , there was no trace of growth of actinomycetes and bacteria on respective Schiff's based agar plates.Vegetables in a spoiled state constitute ethanol oxidizing actinomycetes, yeast & molds, yeast, and bacteria population of 3.91±0.12,6.21±0.01,6.88±0.01 and 7.46±0.15logcfug -1 respectively (Tab 3).Reduction, oxidation and various enzyme activities were higher in dairy sludge(Frąc et al., 2017).

Dyrda et al., 2019; Chatterjee et al., 2006). Statistical Comparison between total microbial and total ethanol oxidizing microbial population in sugar-rich organic waste resources
Initially, the sample characteristics were checked for normal distribution before performing a parametric or non-parametric test.The Kolmogorov-Smirnov and Shapiro-Wilk test reported that the significant values of both total microbial population (TMP) and ethanol oxidizing microbial population (EOM`s) were less than 0.05 (p<0.05),indicating that the measures were not normally distributed.The differences between TMP and EOM`s of spoiled fruits, fruit peel waste, spoiled vegetables, raw and composed sludge of the dairy industry and cane molasses spill were tested hypothetically by Wilcoxon signed rank test (Tab 4).

Table 4
Relation between total microbial population and ethanol oxidizing microbial population of sugar-rich organic waste resources hypothetically tested by Wilcoxon signed rank test Difference

between total microbial population and ethanol oxidizing microbial population of sugar-rich organic wastes Wilcoxon signed rank test statistics
Legend: Confidence level: P<0.05; r -Effect size

Wei et al., 2017; Mokhtari et al., 2012)
(Keffala et al., 2017)ari et al., 2012)fferent fruit samples.Metschnikowia pulcherrima, Aureobasidium pullulans, and Hanseniaspora uvarum were the three most prevalent yeast species in apple (.H. opuntiae was found in sweet lime and fresh fruits of pineapple, watermelon, mango, banana, and orange were found to contain Candida albicans, Saccharomyces cerevisiae, S. ellipsoideus, and Kloeckera apiculata(Gbeminiyi, 2022;Mokhtari et al., 2012).Issatchenkia orientalis, C. tropicalis, C. viswanathii, and Pichia guilliermondii were identified in Sapota, and C. parapsilosis, I. orientalis were found in wine grapes (Rao et al., 2008).fermentans,andGeotrichumcandidum(Mokhtarietal., 2012; Spencer et al., 1992).The current study spotted that the five species of Pichia, as well as the Candida sp.Hanseniaspora sp. and Lachancea sp. of spoiled fruits such as mandarin, banana, sapota, grapes, apple, cantaloupe, and pomegranate, grow in presence of ethanol and functionally reduce ethanol to acetaldehyde (Tab 5).Hanseniaspora uvarum and Pichia kudriavzevii were isolated from pomegranate peels by Utama et al. (2022).One of the possible causes of Pomegranate spots and spoiled symptoms was Candida freyschussii (Patel et al., 2021).Peng et al. (2018)recovered Hanseniaspora opuntiae and Meyerozyma guilliermondii from the peel and pulp of mandarin fruit using an enrichment process.The fruit peels of pomegranate, cantaloupe, and sweet lime (mosambi) fermented under favorable fermentation conditions yielded two species of Pichia and genera Torulaspora sp. and Debaryomyces sp, able to oxidize ethanol.Microorganisms naturally found in vegetables and develop rot under ambient conditions such as aeration, pH, temperature, and moisture.The yeast strains obtained from spoiled fermented vegetables were Saccharomyces bayanus and S. unisporus(Savard et al., 2002).Microorganisms, particularly yeast (Saccharomyces cerevisiae) and molds (Rhizopus stolonifer, Alternaria alternata, Aspergillus niger), cause onion bulb rot.Mold, Aspergillus niger, had the greatest percentage distribution in decaying onion bulbs, whereas yeast, Saccharomyces cerevisiae, had the lowest (Samuel and Ifeanyi, 2015).The deterioration of grated raw carrots was caused by Candida parapsilosis, C. tropicalis, and C. sake(Babic et al., 1992).Geotrichum candidum has been discovered as the cause of watery rot and soft rot on a carrot, cucumbers, tomatoes, and pumpkins (Kim et al., 2011).Tomato spoilage was caused by the life processes of bacteria, yeast, and molds.Microorganisms consume the sugars in vegetables quickly and convert them to acetic acid, lactic acid, alcohol, and carbon dioxide (Ogunniyi and Oladeojo, 2011).The isolates from freshly grated beetroots were dominated by Gramnegative bacteria and yeasts, with no signs of molds (Lopez Osornio and Chaves, 1997).The current investigation established that two species of Pichia and the genus Debaryomyces sp.retrieved from spoiled vegetable waste including peas, bulb onion, small onion, tomato, carrot, potato, beetroot, and radish had the potential to oxidize ethanol.Candida albicans, C. krusei, Lactobacillus plantarum, and L. casei were identified from the dairy plant's activated sludge pond(Keffala et al., 2017).Trichosporon loubieri, Geotrichum sp., and T.

Table 5
Morphological Characterization of Yeasts

view of yeast isolates Colony morphology Microscopic observation Genus identification Texture Color Surface Elevation Margin Cell Shape Cell size (µm) Mode of reproduction Hyphae formation Germ tube formation
Legend: NF -No formation; ±SD; FYES1-8 represents yeast strain from spoiled fruits; FPYES1-4 represents yeast strain from fruit peel waste; VYES1-3 represents yeast strain from spoiled vegetables; CSYES1&2 represents yeast strain from

composed sludge Physiological and biochemical characteristics of ethanol oxidizing yeast from sugar-rich organic waste resources A
Carbohydrate fermentation test was used to study the ability of the yeast strain to ferment sugars in the medium (Tab 6).Pichia species ferment all the tested sugars, glucose, sucrose, maltose, lactose, fructose, arabinose, and xylose and few of the species do not ferment all sugars.Candida species ferment only glucose, sucrose, and fructose among tested sugars.Debaryomyces species ferment glucose, sucrose, and fructose.Hanseniaspora sp.ferments only glucose and fructose.Lachancea sp.ferment glucose, sucrose and fructose.Torulaspora sp.ferment glucose, sucrose and fructose.Magnusiomyces sp.ferments all tested sugars except lactose.Since there is no reversible reaction during testing, the absorption of carbon and nitrogen sources is the primary test for yeast characterization rather than carbohydrate fermentation.Carbon, nitrogen, and vitamin ingestion as the sole source for the metabolic process were examined in an assimilation test.If the test was positive, the analyte remained positive, making the assimilation test more useful in both fermentative and non-fermentative yeast (

Guaragnella and Bettiga, 2021, Palma et al., 2018).
Osmotic pressure kills cells, thus isolated yeast strains that could resist osmotic pressure were examined using an osmotic pressure tolerance test under high concentrations of glucose and sodium chloride.Sugar and NaCl had osmotic responses that were comparable to water activity, but NaCl is harmful because K + may replace Na + in biomolecules, which causes the cells into detoxification mode (

Table 6
Physiological and biochemical characteristics of Yeast Physiological

Table 7
Qualitative analysis of ADH and ALDH activity of yeast strains