Improvement of yeast strain on their stress tolerance

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Abstract

Due to various environmental reasons and depletion of fossil fuels, efforts have been directed to finding alternative fuel resources which are more environmental-friendly, such as ethanol. Currently lignocellulose is preferred for ethanol production as it is derived from biomasses, which are environmentally sustainable, renewable and relatively cheap resource, and causes less environmental pollution. However, the complex degradation of lignocellulose to its monomeric sugars is found to produce inhibitory compounds which will cause a longer lag phase for the yeast cells and inhibit their growth or lower their yield of ethanol production. The lack of a microorganism that is able to fully utilize the 2 major sugar constituents (xylose and glucose) formed by the degradation of lignocellulose is also limiting the use of lignocellulose in this ethanol production industry. Thus the aim of this project is to develop a yeast strain with improved stress tolerance (inhibitors and ethanol tolerance) and the ability to utilize the 2 main sugar groups from the degradation of lignocellulose. Through UV mutagenesis we managed to improve the inhibitor tolerance of the TJU yeast strains. We also managed to apply protoplast fusion successfully to fuse yeast strains Pichia stipitis and Saccharomyces cerevisiae 24860 to obtain fusants with xylose-utilizing ability and higher ethanol tolerance.

Content

Page no.

Acknowlegdements

Declaration

Abstract

Introduction

Materials and Methods

Materials

2.1.1 Yeast strains and culture media

2.1.2 Washing buffers

2.1.3 Selective medias

2.2 Methods

2.2.1 UV Mutagenesis

2.2.1.1 Inhibitor tolerance test

2.2.1.2 Optimization of UV timing / obtaining lethal curve

2.2.1.3 UV-mutagenesis at optimum time to improve inhibitor tolerance

2.2.1.4 Secondary screening of mutant strains

2.2.2 Protoplast fusion

2.2.2.1 Protoplast preparation

2.2.2.2 Genome Shuffling

2.2.2.3 Screening of fusants on selective mediums

Results and Discussion

3.1 UV Mutagenesis

3.1.1 Inhibitor tolerance test

3.1.2 Optimization of UV timing for TJU yeast strain

3.1.3 UV Mutagenesis of TJU yeast strains under optimal timing

3.1.4 Secondary screening of TJU mutants

3.2 Protoplast fusion

3.2.1 Preparation of protoplast

3.2.2 Genome shuffling via fusion of protoplasts

3.2.3 Screening of fusants

3.2.4 Secondary screening of protoplast fusants

Conclusion

References

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1 Introduction

The demand for fuels has greatly increased over the years as we continue to advance. Fuels are greatly used in industrialization and for transportation. Thus, it leads to oil crisis where fossil fuels are depleting at an alarming rate and also caused environmental problems since processing of fossil fuels results in the production of air pollutants. With about 54 % increase in world energy consumption is predicted to occur within 2001 and 2025. Thus, there is an urgent need to find alternative resources and research on this had been and is still ongoing to find a more suitable alternative to replace fossil fuels.

One finding would be using of bioethanol to replace the usage of fossil fuels. The use of bioethanol as a biofuel has increased greatly, as it can be derived from renewable resources such as biomass, which is able to provide substantial amount of energy for the whole world and not causing much environmental pollutions (Zaldivar et al, 2001).

Ethanol production is usually crop-based, utilizing substrates such as sugar cane and cornstarch. However, recently, ethanol production applications have been focused on the utilization of lignocellulose, which are a potentially cheap and abundant polymer found as waste from agricultural, industrial, forestry and municipal solid etc. Also, it is predicted that lignocellulose make up about 50 % of the biomass in the world (Rudolf et al, 2008). It is also environmentally sustainable and will not cause any net increase in carbon dioxide level since the carbon dioxide produced during combustion will be used by plants through photosynthesis, thus resulting in the production of fewer emissions as compared to petroleum fuels (Pasha et al, 2007). With properties of higher octane number and better flammability limits resulting in higher output efficiency, this makes the usage of ethanol as a biofuel more sought after recently (Gupta, 2010).

Conventional ethanol is being derived from grains such as corn after being processed in wet or dry milling method whereby in dry milling, corns are first heat-treated with water and enzyme followed by the addition of a second enzyme which will convert the starch to sugars. The sugars will then be fermented by the yeast to produce ethanol and other by-product such as carbon dioxide. For wet milling, the different components in corn will first be separated before fermenting into ethanol.

Lignocellulose, unlike sugar-containing crops, has a much more complex structure, composing of cellulose, hemicellulose and lignin which form complex called polysaccharides. The breaking down of these three fractions consists of 3 steps; (1) pre-treatment of cellulose and hemicellulose, (2) hydrolysis of cellulose and hemicellulose to release free sugars (pentoses and hexoses) and (3) the fermentation of the sugars to produce ethanol. The whole process results in the release of sugar residues such as hexoses, pentoses and uronic acids. Glucose and xylose comprises two of the largest sugars formed. As a result, for a higher yield of ethanol production from lignocellulose, both glucose and xylose will need to be fermented (Zaldivar et al, 2001).

Unfortunately, the lack of a single microorganism that is able to efficiently ferment all the sugars discharged by the hydrolysis of lignocellulose is limiting the utilization of lignocellulose for ethanol production (Zaldivar et al, 2001).

Degradation of hemicellulose to its monomeric sugars prior to hydrolysis and fermentation will result in the production of inhibitory compounds (Klinke HB et al, 2004). Inhibitory compounds will affect the performances of yeast fermentation and the two main groups of inhibitors produced during pre-treatment are the furans derivatives and weak acids (Palmqvist E et al, 2000). Therefore, it is important to understand more about these inhibitors and the mechanisms of microorganisms which are able to tolerate such inhibitors.

2-furaldehyde, also known as furfural, and 5-hydroxymethyl-2-furaldehyde (HMF) are examples of furan derivatives which are derived from dehydration of pentose and hexoses, respectively (Lewkowski 2001). Both HMF and furfural are known to be the most potent inhibitors out of the many hundreds of inhibitors being produced. The inhibitory effects of furfural and HMF include causing a longer lag phase for the yeast cells and inhibiting their growth or lowering their yield of ethanol production depending on the concentration of the furans and the yeast strain used. HMF and furfural damages the yeast DNA and prevents the synthesis of RNA and proteins (Modig et al. 2002). Both the furan derivatives are shown to directly inhibit the action of alcohol dehydrogenase which plays an important role in fermentation. Moreover, furfural also causes the accumulation of reactive oxygen species and thus, causing actin, chromatin, mitochondrial and vacuole damage in S.cerevisiae (Almeida et al, 2007). The amount of furfural found is usually lower compared to HMF. However, it is still present in sufficient concentration of around 1 g/L to initiate an inhibitory effect on the yeast cells.

No growth of S.cerevisiae has been observed when furfural and HMF is present in the culture medium. When equal concentration of each inhibitor was being compared, it is showed that furfural has a more severe effect as compared to HMF (Taherzadeh MJ et al, 2000).

De-acetylation of hemicelluloses will give rise to acetic acid (Dunlop AP, 1948). While breaking down of HMF and/or when furfural is exposed to acidic conditions at elevated temperature, both will give rise to the release of formic acids and both acetic and formic acids are known as weak acids (Ulbricht RJ, 1984). Weak acids will lower the pH value, thus causing the depletion of ATP which in turn hinders the process of ethanol production (Jeffrey et al, 2005). Due to higher intracellular pH, the acids are able to diffuse from fermentation medium across plasma membrane and dissociate, leading to decreasing levels of cytosolic pH in the yeast cell. Lower cytosolic pH causes the ATPase on the plasma membrane of the cells to pump protons out of the cells which in turn results in lesser amounts of ATP to be used for hydrolysis. This leads to lesser biomass formation. Increased concentrations of these inhibitors will also lead to lower ethanol production.

A study by (Martin et al, 2002) shows that some yeast strains are able to reduce these inhibitory compounds to other compounds. The acids were not converted to a large extent; however, the furaldehydes were reduced to alcohols. However, if the inhibitors are present at high concentrations, no growth and no reduction of the furaldehydes can occur.

Hence, for maximum yield of ethanol productivity from the lignocellulose, a microorganism which is able to tolerate high concentration of inhibitors is necessary (Almeida et al, 2007). This could be achieved by improving the inhibitor tolerance of currently available yeast strains through UV mutagenesis which will allow induced mutations in the yeast cells. Another method would be protoplast fusion. The fusion of the protoplasts of two or more yeast strains together, especially mutant yeast strains with improved inhibitor tolerance, allows the exchange of desirable genetic materials, thus resulting in fusants which possess better inhibitor tolerance.

Currently, there is no single microorganism that is able to fully utilize all the sugar residues constituents of lignocellulose. S.cerevisiae is by far the most outstanding yeast strain for its high ethanol productivity and high ethanol tolerance compared to other yeast strains and it is also recyclable (Shi et al, 2008). When compared with bacteria such as E.coli, S.cerevisiae is shown to have better tolerance towards inhibitors that are being produced during the process of pre-treatment (hemicelluloses to monomeric sugars). However, it is still not an efficient strain as it is unable to utilize xylose, which is the second largest sugar component of hemicelluloses, as it lacks the xylose-utilizing gene.

While there are some yeast strains and bacteria which are able to ferment xylose such as Pichia stipitis, they have either low ethanol tolerance or low ethanol producitivity and thus, not suitable for industrial ethanol production. Thus, efforts have been focused to improve S.cerevisiae by inserting xylose-utilizing genes into the yeast cells however, this method is time consuming and expensive (Almeida et al, 2007).

There are many different techniques being used to culture two different yeast strains (Jeffries, 2000). In co-cultivation, xylose-utilizing yeast and yeast that ferments glucose will be chosen to be grown under the same medium which contains a mixture of sugars. The glucose-ultilizing yeast strain that is most commonly used is S.cerevisiae. In two-stage culture, glucose-utilizing strain will first be cultured followed by the addition of xylose-utilizing yeast strains when the glucose level in the medium had been depleted. However, for sequential culture, the difference is that after glucose supply is depleted, the glucose-utilizing yeast will be removed first before the addition of xylose-utilizing yeast.

Since the optimal growth condition for each yeast strain is different, it will be hard to get the best efficiency out of co-cultivation, and it might even lead to the decrease in the efficiency and product produced.

The conditions for glucose fermentation and xylose fermentation to occur also differ since glucose fermentation requires anaerobic conditions while xylose fermentation occurs only in micro-aerobic conditions. Xylose-utilizing yeast is very much dependent on the oxygen present in the medium to determine the amount of product it can form. Competition between xylose-utilizing yeast strains and glucose utilizing yeast strains will cause lower yield of ethanol being produced. Thus, co-culture is not an ideal way to achieve high yields of ethanol. One possible solution to this would be, to obtain a yeast strain which is able to ferment both glucose and xylose simultaneously through protoplast fusion.

Most xylose-utilizing yeast strains do not have high level of ethanol tolerance and the ethanol tolerance is mostly around 30 to 35 g/l. This will thus, prevents the yeast from fermenting when the ethanol being produced has reached the maximum tolerance level. The solution to it could only be, by using a xylose-utilizing yeast strain which possess high tolerance towards ethanol, or by mutating the xylose-utilizing strains to increase the ethanol tolerance level, or through protoplast fusion of a xylose-utilizing strain and another high ethanol tolerant yeast strain to obtain higher ethanol tolerance yeast which could also ferment xylose.

Recently, a new method, genome shuffling, has caused a huge advance in the construction of mutants with significantly improved phenotypes. Genome shuffling allows the exchange of genes between 2 or more parental strains, thus useful genes can be transferred from one species to another (Verma N et al.).

Genome shuffling had been done on E.coli and the results showed attempts to increase the ethanol tolerance levels of E.coli being successful. The mutants after having increased in ethanol tolerance in the first gene transfer then went through another three transfer before being spread on media to be retained (Conway et al., 1987).

Protoplasts are cells which have their cell walls removed, exposing the cytoplasmic membrane through the means of using enzyme such as snail enzyme and lytic enzyme. The strains after having their cell walls being removed can be examined under microscope where a change in the cell morphologies from being spherical to being circular in shape is observed, or by using the methyline blue staining.

Protoplast fusion is two or more cells having the cell walls removed and cells will then be adhered to each other and fused non-sexually. Through protoplast fusion, parental strains with desired phenotypic improvements are allowed to be recombined where the cells exchange genetic information by fusing into one single cell (Petri et al, 2004). Cells after having gone through protoplast fusion might be able to achieve improved inhibitor tolerance, increased ethanol tolerance and production. Through the repetition of fusion cycles, a library of shuffled strains can be achieved (Shi DJ et al, 2008). Protoplast fusion is a better method in achieving the exchange of better genetic information into another cell, compared to the method of transferring and passing down of better genes through sexual processes.

There are generally two main groups of protoplast fusion - spontaneous and induced fusion. Spontaneous fusion method is when two cells fuse together through plasmodesmata, forming multinucleated protoplasts. However, protoplasts do not usually fuse together due to the negative charges on the surface which cause them to repel. Thus, in order for protoplast to fuse together, it would require the help of either chemical agents or mechanical means which are known as induced fusion. Induced fusion allows faster and easier formation of protoplast fusion as compared to spontaneous fusion.

However, one minus about protoplast fusion is that the genetic exchange is entirely random and sometimes, not all the desired genes get transferred. So it may have to be done repeatedly to achieve good results.

Research had then been directed to apply this method to fuse S.cerevisiae with other xylose-utilizing yeast strains, such as Pichia stipitis. Results from the fusion have shown that the fusants are able to utilize xylose and shows slight improvements on their ethanol tolerance.

Hence, by applying the latest technology of genome shuffling/protoplast fusion, the development of a yeast strain with increased stress tolerance and higher ethanol productivity may be achieved (Zaldivar et al, 2001).

The aim of our research is to improve the inhibitor tolerance of S.cerevisiae strain through UV mutagenesis and protoplast fusion. First, the optimum UV timing is obtained. The selected strain will then undergo UV mutagenesis to improve the inhibitor tolerance. After a few rounds of UV mutagenesis, the strains with the best inhibitor tolerance will be chosen to undergo protoplast fusion to further increase their inhibitor tolerance. The other aim of the research is to obtain a yeast strain which is able to utilize xylose and possess high ethanol tolerance by protoplast fusion. S.cerevisiae and P.stipitis will undergo protoplast formation followed by UV mutagenesis to improve ethanol tolerance. Then both will be fused together and the xylose-utilizing gene from P.stipitis will be transferred over to S.cerevisiae. Fusants will then be screened for the ethanol tolerance and grown on medium containing xylose to ensure the fusants are able to utilize xylose.

2. Materials and Methods

2.1 Materials

2.1.1 Yeast strains and culture media

Two yeast strains; glucose-utilizing yeast Saccharomyces cerevisiae 24860 and xylose-utilizing yeast Pichia stipitis, have been selected and chosen as the starting strains for ethanol tolerance improvement. TJU yeast strains were used as starting strains for inhibitor tolerance improvement. All yeast strains were grown and cultured in YPD liquid mediums [containing 1 % (w/v) yeast extract, 2 % (w/v) peptone and 2 % (w/v) glucose], and incubated at 30 °C, and maintained in YPD solid medium [2 % (w/v) agar], inc ubated at 30 °C and stored at -4 °C. TJU yeast strains were chosen to undergo UV Mutagenesis and protoplast fusion, while S.cerevisiae 24860 and Pichia were chosen for protoplast fusion only.

2.1.2 Washing buffers

NaCl solution [containing 0.9 % NaCl] was used for the washing step in UV mutagenesis. Protoplast formation buffer (PB buffer) [consisting of 20 mM MgCl2, 0.01 M Tris-HCl, pH 6.8 and 0.5 M sucrose] was used as a washing buffer in protoplast fusion and it is also used as a buffer medium to be supplemented with reagents such as 0.01 M Dithiothreitol (DTT), 2 % (m/v) snail enzyme and Polyethylene glycol 6000 (PEG 6000) to be used in protoplast fusion.

Selective medias

YPD liquid and solid mediums supplemented with inhibitor cocktail consisting of Formic Acid, Acetic Acid, Furural and 5-hydroxymethylfurfural (HMF) (Table 1) were used to select inhibitor tolerant TJU yeast mutants derived from successful UV mutagenesis. YNBXE [consisting of 0.67 % (w/v) Yeast Nitrogen Base (YNB), 5 % (w/v) xylose and supplemented with 8 %, 10 %, 12 % (v/v) ethanol] liquid and solid mediums (containing 2 % agar) with and without amino acids were used to select ethanol tolerant and xylose-utilizing yeast fusants derived from successful protoplast fusions.

Table 1 Amount of each inhibitor added for the respective inhibitor concentrations

Inhibitors /concentration(100ml)

45%

48%

50%

52%

Formic acid (ml)

0.1328

0.1416

0.1475

0.1534

Acetic acid(ml)

0.1931

0.2059

0.2145

0.2231

Furural (ml)

0.1143

0.1219

0.1270

0.1321

HMF (ml)

0.1386

0.1478

0.1540

0.1602

2.2 Methods

2.2.1 UV Mutagenesis

2.2.1.1 Inhibitor tolerance test

To determine the maximum inhibitor tolerance for normal TJU yeast strains, TJU yeast strains were inoculated onto YPD solid mediums supplemented with different inhibitor cocktail concentrations and incubated at 30 °C for 2 - 3 days. The highest inhibitor concentration which possess TJU colonies were taken as the maximum inhibitor tolerance for TJU yeast strains.

2.2.1.2 Optimization of UV timing / obtaining lethal curve

To determine the optimal timing for UV mutagenesis, TJU yeast cells were grown in YPD growth medium for 24 hours (to reach mid-log phase). Cells were then washed and re-suspended in 0.9 % NaCl solution. The number of cells were quantified under a microscope and diluted (if needed) to achieve 108 cells per ml. Cells were then divided into 12 small petri dishes (1 ml each) and mutagenized under UV light for 0, 5, 10, 20, 30, 40, 50, 60, 120, 180, 240 and 300 seconds respectively. The UV-treated cells were then incubated in the dark for 2½ hour, to prevent photo-activation repair. Serial dilution was performed to obtain 103, 104 and 105 cells per ml each and inoculated onto 2 YPD plates each. The plates were incubated at 30 °C for 2 - 3 days until colonies were observed. The number of visible colonies were counted and the data is used to plot the lethal curve graph using Microsoft excel.

2.2.1.3 UV-mutagenesis at optimum time to improve inhibitor tolerance

TJU yeast strains were cultured in YPD liquid mediums and incubated at 30 °C for 24 hours. Cells were then centrifuged at 6000 rpm for 5 minutes at 4 °C and washed with 0.9 % NaCl solution. Cells were counted under a microscope, and diluted (if needed) to obtain 108 cells per ml. After which, cells were subjected to UV light for 30 seconds (optimum time) to allow random induced mutations and incubated in the dark for 2 - 4 hours to prevent photo-reactivation repair. After incubation, the mutants were then inoculated onto YPD solid mediums supplemented with different concentrations of inhibitors cocktail (45 % - 60 %) and incubated at 30 °C in the dark, for 3 - 4 days before screening of positive colonies were done. A TJU yeast strain not treated with UV will be used as a control for this experiment.

2.2.1.4 Secondary screening of mutant strains

Mutant colonies appearing on YPD solid mediums supplemented with inhibitors were selected and picked for secondary screening in YPD liquid mediums supplemented with the same or lower inhibitor cocktail concentrations. Cells were incubated at 30 °C for a few days. A TJU yeast strain which was not exposed to UV will be used as a control. Upon observance of growth, selected mediums containing the mutant strains were subjected for another round of UV mutagenesis.

2.2.2 Protoplast fusion

2.2.2.1 Protoplast preparation

Selected Pichia stipitis and S.cerevisiae 24860 yeast strains were cultured in YPD liquid mediums and washed twice with Protoplast formation buffer (PB buffer). Cells were then incubated in PB buffer containing 0.01 M DTT for 30 minutes at 30 °C.

Cells were then collected and washed thrice with PB buffer and re-suspended in PB buffer containing 2 % (w/v) snail enzyme, to allow enzymatic digestion of the cell walls. The cell suspension was incubated overnight at 100 rpm, 30 °C to allow complete digestion of cell walls and formation of protoplasts. Efficiency of protoplast formation was determined by microscopy, by observing the change in the morphology of the cells from a spherical shape to a circular shape. Protoplast fusion can be conducted once 90 % of the protoplasts are formed.

2.2.2.2 Genome Shuffling

After digestion, protoplasts were washed with PB buffer thrice. Protoplasts of S.cerevisiae 24860 and Pichia were then mixed into a tube. The protoplast mixtures were then divided into two equal parts. One part was inactivated with UV light for 10 minutes and incubated in the dark for 2 hours. The other part was heat-treated at 60 °C for 30 minutes. Both heat-treated and UV-inactivated parts were then mixed, and centrifuged at 6000 rpm, 5 minutes at 4 °C. Cell pellet was re-suspended in PB buffer containing PEG 6000 [10 %, 15 %, 20 %, 25 % (v/v) PEG 6000 and 0.01 M CaCl2] to allow fusion of protoplasts. Cell suspensions were incubated and shaken gently at 30 °C for 30 minutes. Fusion efficiencies were monitored under microscope every 10 minutes. After fusion, cells were washed thrice with PB buffer.

2.2.2.3 Screening of fusants on selective mediums

Pichia and S.cerevisiae 24860 fusants were inoculated onto YNBXE (with and without amino acids) solid and liquid mediums [5 % xylose, 8 %, 10 %, 12 % ethanol] and incubated at 30 °C for a few days. Colonies appearing on YNBXE (without amino acids) were selected for secondary screening in YNBXE liquid mediums where their absorbance was recorded at 600 nm to determine their biomass growth.

3. Results & Discussion

3.1 UV Mutagenesis

3.1.1 Inhibitor tolerance test

inhibitor tolerance test_tju.jpg

Figure 1 Wild-type TJU yeast strain on YPD solid medium supplemented with 29% inhibitor cocktail concentration.

Normal TJU strains were inoculated onto YPD solid mediums supplemented with different concentrations of inhibitor cocktail to determine the maximum inhibitor tolerance for this strain. For the first trial, normal TJU strains were inoculated onto mediums containing 15 %, 20 % and 30 % of inhibitor concentrations, with positive results on 15% and 20% inhibitor plates only. For the second trial, the inhibitor concentrations being focused on were between ranges 21 % and 29 %. From Figure 1, we concluded that the maximum inhibitor tolerance for a normal TJU strain is 29 % since it still can grow on 29 % but not further.

3.1.2 Optimization of UV timing for TJU yeast strain

Optimization of the UV timing for UV mutagenesis is important as it determines the UV efficiency. To determine the optimal UV timing to be used during UV mutagenesis for TJU yeast strains, UV mutagenesis was carried out on wild-type TJU yeast strains over a period of 0 - 300 seconds. When compared to the control (0 seconds), there was a significant decrease in the number of colony count as the UV timing increases, as shown in table 2.

Time (s)

Number of colonies

0

72

5

56

10

46

20

31

30

15

40

2

50

1

60

1

120

1

180

0

240

0

300

0

Table 2 Number of TJU yeast colonies appearing on YPD plates after mutation under UV light at different timings. Experiment was done in duplicates, and results were taken from the average.

Figure 2 UV mutagenesis lethal curve for TJU yeast strains, conducted from 0s to 300s.

A drastic drop in the colony count was observed as the UV timing increases, with 30 seconds showing a colony count that differs 80% from the control, as shown in Figure 2. UV timings under 30 seconds resulted in colony counts that are too big indicating that the UV does not affect much of the cells while UV timings above 30 seconds resulted in colony counts that are too little or no colony. This could imply that UV timings after 30 seconds would kill most or all of the cells and there would not be enough cells to further the experiment. Thus, we concluded that the optimum UV timing to be used during UV mutagenesis for TJU yeast strains was 30 seconds as the amount of cells is sufficient and not too much to continue the experiment.

3.1.3 UV Mutagenesis of TJU yeast strains under optimal timing

Normal TJU cells would not be able to grow in YPD mediums supplemented with inhibitor cocktail of concentrations 30% and above as they possess a maximum inhibitor tolerance of 29% (Figure 1). However, after several UV mutagenesis trials, positive colonies were observed on YPD solid mediums supplemented with inhibitor cocktail of 50% in concentration (Figure 3).

Tju(50%-30s).jpgTju(50%-60s).jpg

Figure 3 Colonies appearing on 50% inhibitor plates after UV mutagenesis. UV mutagenesis was done on normal TJU yeast strains to improve the inhibitor tolerance through mutation.

A total of 8 colonies were selected and isolated, to undergo secondary screening in YPD liquid mediums supplemented with inhibitor cocktail and the subsequent selected mutant strains were subjected to a second and third round of UV mutagenesis.

3.1.4 Secondary screening of TJU mutants

Selected mutants were cultured in YPD liquid mediums supplemented with different concentrations of inhibitor cocktail to determine their growth stability. Mutant strains that exhibits growth on the selective liquid mediums were chosen to undergo a second and third round of UV mutagenesis.

All 8 colonies exhibits growth on YPD liquid mediums supplemented with 45% inhibitor cocktail and thus they were selected for a second and third round of UV mutagenesis.

Third round of UV mutagenesis results in mutant strains that can tolerate up to 48% of inhibitor concentration. Sixteen mutant strains were then chosen for a fourth round of UV mutagenesis. Out of the 16 selected strains, only 12 strains exhibited growth on YPD solid mediums supplemented with 54 % inhibitor cocktail (Figure 4).

26052010 54% 3.4.jpg26052010 54% 5.6.jpg26052010 54% 7.8.jpgcolony 9&10 -2.jpgcolony 11&12 -2.jpgcolony 13&14 -2.jpg

Figure 4 Colonies after fourth round of UV mutagenesis appearing on 54% inhibitor plates.

The 12 strains were inoculated into YPD liquid mediums containing inhibitors concentrations of 52 % and 54 % to determine their growth stability. The mutant strains are only able to regenerate in YPD liquid mediums containing 52 % inhibitors cocktail (Figure 5).

Strains 6, 7, 8

Control

Control

Control

Control

Strains 9, 10, 11

Strains 12, 13, 14

Strains 3, 4, 5TN_01CAFAE58B007C009676665327030DAB0.jpgTN_01CAFAE5031A7200964C1FD027030DA80.jpgTN_01CAFAE5175E6F00965A375127030DA90.jpgTN_01CAFAE53067200096684ED227030DAA0.jpg

Figure 5 Regeneration of mutant strains in YPD (52 % inhibitors) liquid medium compared to the

control (normal TJU strain).

Figure 6 Comparison of inhibitor tolerance between normal TJU strain and mutant TJU strains from different rounds of UV mutagenesis

Through several rounds of UV mutagenesis under the optimum UV timing, a significant increase in the inhibitor tolerance was observed when compared to the normal TJU yeast strain (Figure 6). This indicates that the UV mutagenesis process was successful in improving the yeast strain inhibitor tolerance. During the process, the mutant strain experience a slight drop in inhibitor tolerance during the 2nd UV mutagenesis. The reason could be the genes responsible for inhibitor tolerance have been mutated such that the inhibitor tolerance became worst. As UV mutagenesis is random, there will also be chance that it will have the reverse effect from what we expected. However, after several rounds of mutation, an increase in overall inhibitor tolerance is indeed observed.

3.2 Protoplast fusion

3.2.1 Preparation of protoplast

Protoplast formations were achieved by the addition of snail enzyme (snailase) to digest the cell walls. We worked with different concentrations of snailase to determine the optimum concentration of snailase used. The efficiency of protoplast formations were greatly affected by the concentration of snailase used and we concluded that 2 % (m/v) snailase is the optimum concentration as concentrations above this may cause cellular breakage.

Efficiency of the protoplast formations were determined by microscopy by observing the changes in cell morphologies. Protoplasts appear in a circular shape. Complete protoplast formations for Pichia stipitis (Figure 7) and S.cerevisiae 24860 (Figure 8) require approximately 24 hours before protoplast fusion can be continued.

26.5hour

0hourpichia_protoplast.jpg Figure 7 Protoplast formation efficiencies using Snail enzyme, viewed under microscope at 40X magnification for Pichia stipitis. Arrows represent protoplasts.

26.5hour

0hoursc_protoplast.jpg

Figure 8 Protoplast formation efficiencies using Snail enzyme, viewed under microscope at 40X magnification for S.cerevisiae 24860. Arrows represent protoplasts.

3.2.2 Genome shuffling via fusion of protoplasts

Fusion of S.cerevisiae 24860 and Pichia protoplasts were done in PB buffer containing PEG 6000 and CaCl2. Optimization of the PEG 6000 concentration is done using different concentrations of PEG 6000 (10 %, 15 %, 20 %, 25 % (v/v)) (Figure 9). It is then concluded that PEG 6000 concentration of 20 % is the optimum PEG concentration to be used for fusion as higher concentrations of PEG 6000 would be potentially toxic to the cells. Optimization of fusion timing was also done by carrying out protoplast fusion for different timings. It is concluded that 30 minutes is the optimum timing for fusion as incubation in PEG 6000 longer than 30 minutes would also be toxic to the cells.

Fusion efficiencies were monitored under microscopy every 10 minutes.

Fusion (25% PEG)

Fusion (20% PEG)

Fusion (15% PEG)

Fusion (10% PEG) fusion-3.jpg

Figure 9 Cell fusion efficiencies of S.cerevisiae 24860 and Pichia stipitis viewed under microscopy at 40X magnification at 30 minutes.

3.2.3 Screening of fusants

Controlcontrol.jpg

Figure 10 Pichia and S.cerevisiae 24860 on YNBX (without amino acids) solid medium (control).

Pichia and S.cerevisiae 24860 controls were inoculated onto YNBX solid medium (Figure 10). Figure 10 shows that Pichia exhibits growth on YNBX (5 % xylose) solid medium while S.cerevisiae 24860 does not exhibit any growth at all due to its inability to utilize xylose.

Fusants of Pichia stipitis and S.cerevisiae 24860 were inoculated onto YNBXE solid and liquid mediums (with and without amino acids) with ethanol concentrations of 8%-12% (Figure 11). YNBXE mediums without amino acids lack the essential amino acids needed by yeast cells to grow, thus the yeast cells will only depend on xylose as their carbon source for growth. This selection medium will be used to determine the fusants which are able to tolerate ethanol and utilize xylose simultaneously. Successful fusants appearing on YNBXE selective mediums were subjected to secondary screening.

10% ethanol YNBXE (with amino acids)

10% ethanol YNBXE (without amino acids)

8% ethanol YNBXE (with amino acids)

8% ethanol YNBXE (without amino acids)YNBXE-1.jpg

12% ethanol YNBXE (with amino acids)

12% ethanol YNBXE (without amino acids)ynbxe-3.jpg

Figure 11 Colonies appearing on YNBXE (with and without amino acids) with different ethanol concentrations for S.cerevisiae and Pichia stipitis fusants under aerobic conditions.

Pichia yeast strains cannot tolerate ethanol concentrations of 8% or higher, while S.cerevisiae 24860 yeast strains were unable to grow on mediums containing xylose. Thus, we can conclude that colonies appearing on our YNBXE plates are indeed fusants from successful protoplast fusion since the medium contain 8% or higher ethanol concentration and 5% xylose. From Figure 11, colonies appearing on YNBXE plates (with amino acids) indicate fusants that can tolerate ethanol while colonies appearing on YNBXE plates (without amino acids) indicate fusants that are able to utilize xylose and tolerate ethanol at the same time.

3.2.4 Secondary screening of protoplast fusants

A total of 12 colonies were selected from YNBXE plates (without amino acids) and inoculated into YNBXE liquid mediums containing 8% ethanol and their absorbance readings were recorded.

Figure 12 Comparison of absorbance readings between the fusants and 2 controls (Pichia and S.cerevisiae 24860).

Figure 12 confirmed that the fusant strains 8, 14, 15, 16, 17 and 18 exhibits growth in the selection media when compared to the other fusant strains and the Pichia and S.cerevisiae 24860 controls. This proved that fusant strains 8, 14, 15, 16, 17 and 18 possess higher ethanol tolerance and the ability to utilize xylose, which is the 2 desirable traits of the parental strains; S.cerevisiae 24860 and Pichia stipitis. Thus this indicates that the protoplast fusion process was indeed successful.

4 Conclusion

The complex degradation process of lignocellulose results in the production of inhibitory compounds which will affect the growth of yeast cells or lower their yield of ethanol production. Normal TJU yeast strains are only able to tolerate up to 29 % of these inhibitors cocktail concentration. However, through this study, UV mutagenesis is found to be a good and fast method to induce random mutations in the yeast cells, thus resulting in yeast cells with improved inhibitor tolerance.

Through applying several rounds of UV mutagenesis on normal TJU strains, we managed to obtain TJU mutants strains that possess higher inhibitor tolerance compared to the normal TJU strains. The TJU mutant strains are shown to be able to tolerate inhibitor concentrations of up to 54 % and regenerate on YPD liquid mediums consisting of inhibitor concentrations of up to 52 %. Protoplast fusion on the selected TJU mutant strains is currently ongoing for further work studies on improving the inhibitor tolerance.

Besides inhibitory compounds, degradation of lignocellulose results in the release of glucose and xylose as the 2 major sugar constituents. The yeast strain S.cerevisiae 24860 is currently the most preferred microorganism for ethanol production due to its high ethanol production and high ethanol tolerance, however, it alone is not able to utilize xylose to produce ethanol. However, after protoplast fusion with the xylose-utilizing yeast strain Pichia stipitis, xylose-utilising genes from Pichia get transferred into S.cerevisiae, and the newly formed fusants are shown to possess desirable traits from the two parental yeast strains. The fusants show xylose-utilizing ability and higher ethanol tolerance. This indicates that protoplast fusion is indeed a good powerful breakthrough as it allows the transfer of genes of interest from one organism to another.

For further studies, the fusants will be subjected to fermentations to observe their ethanol productions.

In conclusion, the use of lignocellulose in the bioethanol production industry is believed to provide several benefits to the world and the environment. Apart from economic advantages and reducing environmental problems caused by the burning of fossil fuels, the use of lignocellulose, which is derived from biomasses, can also promise us with sufficient fuel resource to last us for decades. Moreover, with the application of new modern science to improve microorganisms for such industrial use, ethanol production processes from the biomass compounds can be further maximized.

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