Bioethanol Production by Candida tropicalis Isolated from Sheep Dung

: Microorganisms have an active role in biotechnology for example yeasts, especially in some genus like Saccharomyces , Pichia , and Candida . C.tropicalis one of the most important species of Candida and despite it is one of the causative agents of candidiasis but it has a major role in the production of many chemical compounds. C.tropicalis in the previous study was isolated from sheep dung and morphologically and molecularly classified the result of sequencing was elucidate 100% similarity between the studied isolate and other isolates inserted in DNA Data Bank of Japan DDBJ, physiologically this isolate tolerated 6% ethanol concentration in broth media with the ability to the production of 4% ethanol under optimum conditions including (YEPDB) medium, incubation temperature 30◦C, PH 6 and dextrose as a carbon source which give more productivity as a carbon source and optimum incubation period was three days . This isolate gives a positive result for the cellulase enzyme-producing test and an exciting feature to remove effluent because it completely adsorbed the Congo red dye from dye solation in a liquid growth medium.


Introduction:
Bioethanol is the ethanol produced from biological sources such as feedstocks rich in sucrose such as fruit, corn, and sugar cans, lignocellulosic biomass such as wood, micro, and macroalgae biomass by fermenting a wide range of sugars to ethanol 1 . Bioethanol is the most used biofuel worldwide because it reduces crude oil consumption and environmental pollution 2 . Also, microorganisms such as yeasts have an essential role in bioethanol production. There are three essential steps in the generation of bioethanol, the first is an initial test of lignin residues, the second is cellulose and hemicelluloses hydrolysis, and the third is simple sugar fermentation 3,4 . Mainly most yeast miss the ability of degradation of cellulose to simple sugar so that suggested growing of fungi that have this ability like Trichoderma reesei with yeast in the same fermentation medium to produce alcohol 5 .
Candida is a genus with many species, some found as normal flora in human and animal bodies 6 . Candida tropicalis is a common species mainly treated as an opportunistic pathogen, but it is considered a promising tool in biotechnology because of its unique features such as high ability to produce bioethanol, enzymes, and many compounds 7 .
The most common yeasts that produce ethanol are C.tropicalis, Saccharomyces pombe, and S.cerevisiae, the first one has fermentation ability, which resembles the last one 8 . One study suggested that the fungus Trichoderma ressi can be grown in the same fermentation medium to produce alcohol using cellulose. C.tropicalis ferments the starch to ethanol in the little mount, while other strains isolated from herbivore dung can ferments xylose to ethanol with concentrations ranging 1.2-2.3 g/l also produced high concentration of xylitol 9, 10 In cellulose biotechnology, C.tropicalis plays an active role in producing extracellular enzymes like cellulose 11 . Also, yeast single cell protein is produced by the same strain of C.tropicalis by using sugarcane bagasse hemicellulosic hydrolysate as a substrate of a Carbone source 12 . This study aims to evaluate the ability of C.tropicalis isolated from sheep dung to produce ethanol, cellulose enzyme, and adsorption ability.

Material and Methods:
Isolation of Yeast Sheep dung was collected in a clean paper bag and dried at room temperature. Samples were cultured using moist chamber methods in which sterile filter paper was moistened by sterile water in a petri dish and incubated at 30•C for three weeks with the periodic examination 13 .

Identification of Yeast:
Candida CHROM Agar was used for the morphological identification of Yeast, and micro identification is also carried out by compound microscope depending on 14 .

Molecular Identification of Yeast:
The DNA extracted by Presto mini gDNA yeast kit (Bioneer) and amplified using universal primers ITS1: F-5-TCC GTA GGT GAA CCT GCG G-3 and ITS4: R-5-TCC TCC GCT TAT TGA TAT GC-3. Amplification was carried out by using the EconoTaq plus Green Master Mix (Lucingen). The following PCR program was used: 35 cycles, including an initial denaturation at 95 •C for 2 min. Subsequent denaturation was at 95 •C for 30 s, annealing at 50 •C for 30 s, and extension at 72 •C for 1min. A final extension at 72 •C for 10min was followed by holding at 4 •C. Additionally, ITS Gene space was sequenced by forward, and reverse primers in the Macrogen Inc and the data processed by the BioEdit software and the nucleotide sequence was analyzed using the Basic Local Alignment Search Tool (BLAST). The isolate was inserted in DNA Data Bank of Japan DDBJ and take the name 1FRNOJA and the Accession No is LC647056 http://getentry.ddbj.nig.ac.jp/getentry/na/LC647056

Tolerance and Production of Ethanol
Medium (YPDB)composed of 10gm of yeast extract, 10gm of peptone, and 20gm of dextrose used for producing and tolerance ethanol, different concentration of ethanol prepared (1,2,4,6,8,10 )% in broth culture medium and all concentrations are inoculated with isolated C.tropicalis after activation. Flasks were incubated for three days at 30•C. After the incubation period, the absorbance was detected at 600 nm to measure the growth density of Yeast in the medium 15 .

Qualitative Determination Test of Ethanol:
The (YPDB ( medium was inoculated with an activated isolate of C.tropicalis and incubated for three days in 30•C. After the incubation period, the broth medium of cultivation was centrifuged, and the supernatant was distilled. The reagent (0.5mL sulphuric acid H2SO4and 1mL potassium chromate K2Cr2O7 ) was added to 1mL of broth medium, and the mixture was incubated at 60°C for 15 min, and volume was completed to 10 ml with distilled water. Transferring the mixture from orange color to blue-green color indicated the presence of ethanol 9 .

Quantitatively Determination of Ethanol by Standard Curve
Quantitative estimation is carried out by comparing the absorbance of the medium containing ethanol produced by C.tropicalis with different standard curve concentrations in O.D.600 nm 16 .

Adsorption of Dye
Dye adsorption ability of C.tropicalis cells was studied using Yeast grown in SDB medium until OD reached 1.0 and transferred to congo red solution with a final concentration of 20-100ppm. The experiment was conducted in 100ml conical flasks. Dye absorption was investigated by taking OD 497nm intermittently 9 .

Optimization of Ethanol Production
To optimize the production of ethanol by C.tropicals, firstly, different mediums were used ( potato dextrose broth PDB, sabouraud dextrose broth SDB, yeast extract peptone dextrose broth YEPD). Also, different parameters were tested, including incubation temperature, which was determined by growing the C.tropicalis isolate in the most suitable medium at different temperatures (25°, 30°, 37° ). Moreover, optimum pH was investigated by cultivating C.tropicalis in the medium with pH adjusted to 5.0, 6.0, and 7.0 at the optimum temperature. Likewise, the effect of carbon sources (glucose, maltose, sucrose, and dextrose) was also tested. The incubation period also tested (2,3and 4) days. The growth in different conditions was measured intermittently in OD (optical density at 600 nm) 17 .

Cellulase Enzymes Production by C.tropicalis
Mandels and Weber medium (MW, 1969), in which carboxymethyl cellulose (CMC) considers as an essential source of carbon and energy, was used to evaluate the ability of C.tropicalis to produce cellulase enzyme. Medium composed of (KH2PO4, K2HPO4, MgSO4.7H2O, yeast extract, Carboxymethyl cellulose, Agar) C.tropicalis isolate was grown in MW medium and incubated at 30∘C for three days. The cellulase

Isolation of Yeast:
After two weeks, many fungi are grown in the dung as well as Yeast.
The yeast isolate is purified on SDA and maintained in slant on 4•C.

Identification of Yeast Isolate:
The growth of Yeast in SDA was cream-colored and dull smooth colonies after incubating period 3day in 30 •C while the color on Candida CHROM agar was dark blue colored Fig. 1, A

Tolerance and Production of Ethanol
The isolate C.tropicalis tolerated ethanol in the medium it can grow in broth media with 6% ethanol, Table 1.

Qualitative Determination Test of Ethanol :
When potassium dichromate reagent (0.5ml sulphuric acid H2SO4 and 1ml potassium dichromate K2Cr2O7) is added to 1ml of broth medium that contains C.tropicalis are producing ethanol. The blue-green color indicated that ethanol presence on broth media. Fig. 3.

Figure 3. Detection of ethanol in broth media A (blue-green color = reagent + broth medium with ethanol produced by C. tropicalis), B (orange color = reagent + broth medium without growth (control)
Quantitative Determination of Ethanol: This C.tropicalis isolate was found can to produce about 4% V/V of ethanol by comparing the absorption of the ethanol concentration that is found in growing broth medium of C.tropicalis in ( OD600)with the ethanol standard curve. Fig. 4.

Adsorption of Dye
The result showed that the high ability of C.tropicalis isolates to adsorb Congo red dye in broth medium after the incubation period, the dye completely removed from the medium in all concentrations of dye . Fig. 5 A&B. In flask A we can see the yeast cells that adsorbed the dye deposited in the flask bottom.

Optimization of ethanol production:
The result shows that the YEP is the most suitable for producing ethanol at 30 o C and PH 6. Dextrose gives more productivity as a carbon source. The optimum incubation period was three days Cellulase Enzymes Production by C.tropicalis: A clear light halo appeared in the zone of colony growth. This indicates that the CMC in the medium was degraded by the cellulase secreted by C.tropicalis colony Fig. 6.   Figure 6. Production of cellulase on CMC medium by C. tropicalis after incubation for three days at 30ᵒ C appeared as a light halo.

B A
This test revealed that the studied C.tropicalis isolate was non-biofilm forming because the isolate appeared with a pink (wine red) colored colony in CRA medium after incubation Fig.7.

Discussion:
Many yeasts capable of fermenting the sugar and starch media supplement alpha-amylase enzyme to ethanol. One of these yeasts is C.tropicalis that ferments the hexose sugar to bioethanol 5 . C.tropicalis can be isolated from different sources like food samples such as juice and honey 20 . And sugarcane, plant, and garden soil. 5 In a previous study, C.tropicalis was isolated from sheep dunglike wise 21 in which many Candida species were isolated from herbivores dung with the observation of high ability of these Candida isolate to the production of ethanol and xylitol. Initially, the isolate was identified morphologically and molecularly by ITS genetic region because the most widely used molecular targets for identification of yeast are the 28S nuclear ribosomal DNA and internal transcribed spacer region 22 . The studied C.tropicalis isolate exhibited tolerance to 6% ethanol concentration in growth broth medium with significant ability to the production of bioethanol with the amount equivalent to 4% (v/v), and this amount was the approach to 7 . in which C.tropicalis isolate produced about 5.45% v/v of bioethanol and less than that obtained from Saccharomyces cerevisiae RL-11 23 . Bioethanol C2H5OH is either used directly as pure ethanol or mixed with the gasoline to form (gasohol) 2 . In the qualitative assay of ethanol production, the orange color of potassium dichromate reagent transfers to dark green color in the founding of sulfuric acid because of ethanol oxidation to carboxylic acid. The main factor that effecting bioethanol production was culture media. The studying experiment revealed that the optimum medium for ethanol production was YEP with Carbone source Dextrose, and the incubation temperature was 30 o C. This is similar to the fermenting process with C.cerevisiae that was carried out at 30 o C while the optimum pH was 6 because ethanol production is very high in an acid medium. When pH is raised to 9 the productivity of ethanol will be reduced substantially 2 . The dye adsorption ability of studied C.tropicalis isolate was examined by using Congo red dye. The result revealed high adsorption ability and complete removing of Dye from growth broth medium by the yeast cell, which shows this isolate has an exciting feature to remove effluent because adsorption techniques are widely used to remove certain classes of pollutants from water, especially dyes that considers problematic groups because of their ability to pass through conventional treatment system without big change, also some microorganisms like Chlorella vulgaris , Nostoc paludosum and the yeast like S.cerevisiae have dye adsorption ability 24,25 . The cellulase enzyme test was positive to studied C.tropicalis isolate because light clear halo appeared in the zone of colony growth this indicates that the CMC in the medium was degraded by the cellulase secreted by Yeast so that complex carbohydrates (cellulose ) turning into simple sugars especially yeast isolated from sheep dung that central it was founded in the medium that cellulose found in the gut of sheep and enzymatic capacity depending on the environment in which the yeasts found this ability to production cellulase enzyme by this C.tropicalis isolate consider promising result because the yeast can produced bioethanol from cellulose not only from the fermentation of simple surge also cellulase enzyme play role in the treatment of agriculture waste and the bioremediation of cellulatic materials for sustainable bioethanol production 26,27 biofilm test show that isolated was non-biofilm forming because the isolate appear with pink (wine red) colored colony in CRA medium after incubation periods, yeast cells in biofilm survive harsh growth conditions as biofilms are surrounded by high molecular weight extracellular polymeric substances (EPS) that attach cells 28,29 . To elimination of biofilms needs amalgamation of hydrolytic enzymes that can degrade proteins, polysaccharides, eDNA, and QS molecules 30 . Cellulase enzyme succeeds in inhibiting biofilm production in Bacillus sp. by degrading the exopolysaccharide matrix 31 . So that cellulose enzyme prevents biofilm formation in studied C.tropicalis because it was isolated from dung that main it was in medium rich with cellulose and cellulase enzyme in the sheep gut.

Conclusion:
C.tropicalis can be isolated from dung as well as this isolate has good productivity to bioethanol and cellulase enzymes so it can be used successfully in production of bioethanol through fermentation of sample sugar and cellulose due to its ability to secrete cellulase enzyme . The high ability of dye adsorption and complete removing of Dye from aqueous solution by the yeast cell will help in decreasing water pollution by removing effluent because adsorption techniques are widely used to remove pollutants from water.