Hee Yen Zhen
42724614
MICR3003: Molecular Microbiology
Practical: Beer & Biofuels
Abstract
Fermentation is a process where Saccharomyces cerevisiae (yeast) produces ethanol and
biomass. The selected yeasts with specific strains from Wyeast Laboratories and White Lab
collection were chosen in this experiment. The aim of this paper is to culture the four selected
yeast strains (S288C, WLP004, WLP099 and WLP500) and undergone fermentation in yeast
nitrogen base 2% glucose broth. The ethanol (biofuel) production was measured using Gas
Chromatography and the biomasses are recorded using a measuring weight. The specific
traits of the yeasts were determined using bioinformatics approach based on their presence of
ethanol concentration and biomass production. The WLP500 yeast strains were able to
produce the highest amount of ethanol concentration 1.18% (with an average of 1.12%) and
WLP400 yeast strains were able to produce the most biomass, 147 mg/ml (with an average of
137.33 mg/ml). WLP099 were assumed to produce the highest ethanol and biomass
production in this paper but were unsuccessful due to practical handling and human error
during the experiment. In this paper, WLP500 yeast strains were selected to be the best yeast
strains in the production in biofuel and Vegemite for WLP500 were able to produce high
ethanol concentration and about similar biomass as WLP400 strains.
Introduction
Saccharomyces cerevisiae or also known as Brewer’s yeast / Baker’s yeast are single-cell
eukaryotes that play an important role in our daily agriculture and food processing. Yeast can
be traced back 3,000 years where the Egyptians and Babylonians utilised the fermentation of
wild yeast to alveolate the bread and is then later discovered by Louis Pasteur in 1845,
claimed that these microorganisms are capable of fermenting sugar, produce ethanol and
carbon dioxide. Since then, Saccharomyces cerevisiae are industrialized in manufacturing
fermented product. The byproduct of Saccharomyces cerevisiae from fermenting the sugar
were commonly used in alcoholic beverages and fermented products in the industries, and
applicable research in laboratory in searching and providing the most efficient and specific
yeast for the market needs (1, 2, 3).
Biofuel is a type of hydrocarbon solution that consists of ethanol for combustion in the
engine of all the transportation. The amount of ethanol in the biofuel is essential and the
absence of ethanol causes no combustion. Therefore, using yeast to generate biofuel from
sugar is an alternative possibility. It was recently reported that University of Texas
researchers were trying to convert yeast cells into generating biofuels in replacing fossil
fuels(4).
The selected yeasts from the list from Wyeast Laboratories and White Lab collection strains
were WLP500, WLP099, WLP004 and S288C, based on each of their specific genes (ADH1,
PDC1 and CDC19) were selected from the superpathway of glucose fermentation to
determine which genes are responsible for the efficiency of ethanol production. The ADH1
gene is responsible in converting acetaldehyde to ethanol in the last step of fermentation
pathway. The PDC1 gene is one of the pyruvate decarboxylase isoenzymes that convert
pyruvate into acetaldehyde and carbon dioxide during the fermentation before the ADH1
gene. The CDC19 gene is a pyruvate kinase gene that will converts phosphoenopyruvate
(PEP) for both aerobic and anaerobic respiration. Without CDC19 gene, the yeasts were not
able to uptake glucose as the carbon source (5, 6, 7, 8).
The microbial biomass produced from Saccharomyces cerevisiae is a suitable supplement of
protein and vitamin B which were widely used in the commercial health products. The
specific gene (TPK1) that attach a subunit of cAMP-dependant protein kinase A that will
regulates cell growth, stress response and other processes (9, 10).
In this paper, the four selected yeasts’ strains were determined the best for production in
biofuel and Vegemite. The yeast strains that were able to produce high ethanol will produce
less biomass, therefore WLP099 and WLP500 were proposed to produce high ethanol
concentration than the other selected strains.
Materials and Methods
Firstly, the selected yeast namely WLP500, WLP099, WLP004 and S288C were culture into
3 falcon tubes containing 8ml yeast nitrogen base (YNB) 2% glucose each and plated on each
YPD culture plates respectively and the plates were incubated at 18°C. That would be the
starter cultures during that time.
Once the 12 starter cultures had showed growth, each of the 12 tubes was resuspended and
pitched into a 250ml bottle containing 150ml YNB individually. The bottles were labelled
according to the 12 previous falcon tubes. These bottles were then capped with airlocks and
incubated at 21°C for a week. As for the DNA purification, the grown yeast on the culture
plates were then scraped up a large match head worth with a pipette tips and resuspended in
microfuge tube contained 0.5ml TENTs buffer. The yeasts were then added with 0.2ml of
acid wash 0.45mm glass beads and 0.5ml phenol-chloroform and vortex for 20 minutes.
These yeasts were then centrifuged for 10 minutes at max speed which will result a multiple
phase where the top clear aquas phase is extracted and transferred into a new labelled
microfuge tube. 40ul (1/10v) of 3M sodium acetate and 1 ml 100% ethanol is added, then the
tubes were inverted 3 times to mix. The DNA will precipitate at -20°C for about 30 minutes
or more. The tubes were then centrifuged for 15 minutes at max speed. The supernatant of
each tube were decanted and carefully without disrupting the pellets. The pellets were then
washed with 1ml of 70% of ethanol and centrifuged for 1 minute. The supernatant is decanted
and the pellets were left to dry. When the pellets were dried, 150ul TE buffer is added to
dissolve the DNA and stored at -20°C for a week.
Next, when all the bottles had undergone fermentation, they will be tested for Gas
Chromatography (GC) for Ethanol content. The solution in the bottles was shook and about
5ml of the solution were drawn with a syringe. The syringe was then attached with a filter
unit to filter out the solution into a falcon tube and 1.5ml was then pipetted into a 2ml GC
sample vial. These processes were repeated for the rest of the bottles. As for the biomass
measurement, 10ml of the solution was pipetted from the same bottles into a falcon tube
respectively. The tubes were centrifuged and the supernatant were decanted. The samples
mass were then weighted and calculated. As for the Random Amplification of Polymorphic
DNA (RAPD), the isolated DNA from the microfuge tubes were resuspended and 1ul of each
DNA of the yeast strain is pipetted into PCR tubes containing 24ul of PCR Master Mix and
labelled respectively. The primers for this RAPD are M13 and (GACA)4 and were done
separately which consist of 10 PCR tubes. The PCR will undergone 35 cycles and were
stored at 4°C for a week.
Finally, the PCR tubes containing the DNA of the yeasts strains were added with 2ul of
loading dye individually and vortex to mix. There were 2 gels prepared for the different
primers respectively. The first gel is labelled for M13 primer detection. 6ul of 1kb ladder was
loaded to both ends of the wells and the stained yeast strains were pipetted accordingly
(control, S288C, WLP004, WLP099 and WLP500). The process were repeated for the second
gel which is labelled for (GACA)4 detection. The voltage was set to 90V and both gels were
run for 30 minutes. Once the front dye had migrated about ¾ distances away from the wells,
the run was stopped and the gels were placed on a UV transmilluminator to visualise the
bands. The results of the gels were photographed.
As for the bioinformatics approach which was done by a lab partner (Kurt Harris), the
Illumina Data were imported into CLC and the S288C reference genome was downloaded
using CLC. Each genome strains of the yeasts (WLP004, WLP099 and WLP500) were used
to align with the S288C strains. Probabilistic Variation Detection such as finding the variants,
SNPs, MNPs, and InDels were conducted and the total numbers of variants per strain were
recorded. Marker genes were found using genome database and the first three key genes were
selected which involved in the fermentation pathway. The numbers of variants per gene of
each strain were recorded.
Results
Fermentation. All the starter cultures except one of the three WLP099 starter culture did not
grow and the bottle that was prepared for its fermentation was used as a Control. Based on
Table 1, the three bottles of yeast strains S288C did not grew in the media similarly to the
Control. The other bottles for WLP004, WLP099 and WLP500 showed growth and
fermentation in the bottles. There are no bubbles or carbonation occurred in the fermentation
bottles for the media used in the fermentation is YNB 2% glucose.
Biomass Measurement. The absence of fermentation from Control and S288C bottles would
not be measured for their biomass. From Table 2, the biomass for yeast strains WLP004,
WLP500 and WLP099 has been collected and calculated from 10ml samples. The average
biomasses are 13.73 mg/ml, 12.73 mg/ml and 10.8 mg/ml respectively. WLP004 produced
the most biomass, while WLP009 produced the least biomass showed in Table 2.
Gas Chromatography. The Gas Chromatography results obtained were all calculated in
Area (uV.s) within 1.997 to 2.001 minutes timeframe. The Graph 1 has been tabulated based
on the standard ethanol of 2%, 4%, 6%, 8% and 10% in to measure the percentage of ethanol
from 1ml of GC samples and Table 3 has been tabulated. The absence of ethanol in Control
and S288C showed mild production of ethanol. The WLP500 yeast strains produced the
highest ethanol concentration which is about 1.18% individually and an average of 1.12%,
while the WLP099 yeast strains produced the least ethanol concentration which is about
1.06% individually and an average of 10.8%.
Random Amplification of Polymorphic DNA (RAPD). The bands in Figure 1 showed that
the random amplification produced by the primer (GACA) 4. The S288C had dark bands
between 0.5-1.0kb and a light band below 0.5kb. The WLP004 had a dark band between 0.51.0kb. The WLP099 had a dark band below 0.5kb. The WLP500 had a dark band similar to
the bands in WLP004 and S288C, and a light band below 0.5kb.
The bands in Figure 2 showed that the random amplification produced by the primer M13.
All the yeast strains in Figure 2 showed there were 2 dark bands between 0.5-1.0kb. The
288C and WLP099 had an additional dark band at 3.0kb, the WLP099 and WLP500 had an
additional dark band between 2.0-3.0kb, and the WLP004 has a dark band at 2.0kb.
The common bands that showed in all the yeast strains were tend to be the common
consensus region from the yeasts’ strains
Bioinformatics. From the bioinformatics approach, the Saccharomyces cerevisiae [Gene
annotations (638)] was used as the reference genome to compare the yeast strains (Figure 5,
6, 7, 8). The alignment of the yeast strains (S288C, WLP004, WLP009 and WLP500) showed
in Figure 3. The number of variants had been detected from the yeast strains and tabulated in
Table 4. The three key genes (ADH1, PDC1 and CDC19) was selected from the
Saccharomyces cerevisiae fermentation pathway (4) and another gene (TPK1) to determine
the production of biomass has been tabulated in Table 5. The yeast strain, WLP500 had the
highest number of variants compare to the other yeasts’ strains (Table 4). This showed that
WLP500 had variants present in the 4 selected marker genes (Table 5). The yeast strains were
then tabulated in a phylogenetic tree (Figure 3) to determine the likelihood among the yeast
strains. The WLP004 yeast strains from Figure 3 was shown as outgroup and the furthest
distance, 0.026775, between WLP004 clade and S288C clade.
Discussion
The initial of the experiment showed the absence of culture growth in one of the WLP099
falcon tube followed by the absence of fermentation in all S288C bottles. The experiment
was then preceded to GC analysis and biomass measurement with the fermented samples.
From Table 3, the individual bottle of WLP500 produced the highest concentration of
ethanol, 1.18% and average of 1.12% within a week. WLP099 was assumed to produce
higher amount of ethanol than WLP500 based on the information listed from Wyeast
Laboratories and White Labs collection strains (Table 6), but from the results in Table 3,
WLP099 yeast strains weren’t able to produce high ethanol concentration and much lower
than WLP004, which were assumed to be the lowest production of ethanol in this experiment.
The WLP099 biomass were assumed to produce within the range between WLP500 and
WLP004 biomass production based on their flocculation provided in Table 6. However, the
data collected showed in Table 2 showed WLP099 biomass was lower than WLP500 by
19.33mg/ml. These claimed that WLP099 might have undergone practical handling error
which explains the absence of growth in WLP099 starter culture. Furthermore, the WLP099
yeast were unable to execute its full potential during the fermentation might due to the short
timeframe given.
From the bioinformatics approach, the yeast strain S288C was used as the reference yeast
strain of the experiment. In Figure 1, the primer (GACA) 4 was able to fingerprint the
common region of WLP500, WLP004 and S288C yeasts’ strains. This might showed that the
three yeast strains had common properties such as alcohol tolerance or the limitation of
attenuation (Table 6), while the WLP099 band which were lower than 0.5kb, might showed
the properties of high alcohol tolerance or higher attenuation than the other yeast strains. As
Figure 2, the bands produce from the primer M13 showed almost all the bands are common
in all the strain except the density of the bands. The high density bands between 0.5-1.0kb
showed the relativeness of S288C and WLP500 based on ADH1 alignment in Figure 3, but
the band at 3.0kb showed low density which explained the variable of WLP500 in Figure 3,
differed from other strains from position 623-682 bases (purple boxes). On the other hand,
the bands between 2.0-3.0kb are present in S288C, WLP099 (high density) and WLP500
(low density). These might be the band of interest in producing high amount of ethanol,
which the primer M13 replicated the most in WLP099 based on the high density of the band.
The ADH1 alignment (Figure 3) showed at position 925 bases (red box) was Guanine for
WLP099 where others were Adenine. This might conclude that the position 925 bases (red
box) in WLP099 were the specific region for producing high amount of ethanol.
As for CLC Genomic Workbench Approach, WLP500 had the highest number of variants
detected (Table 4) which leads to the present in all selected marker genes (Table 5). This
explained that the results of WLP500 had high ethanol production (Table 3) in this
experiment. The S288C and WLP099 showed lower in both number of variants and marker
genes detected (Table 4 & 5). These claims explained that results of absence growth during
the fermentation process of this experiment.
In conclusion, WLP500 was practically the best selected yeast strain for the production of
ethanol and biomass which was the biofuel and Vegemite for this paper from both analysed
results and the bioinformatics analysis. The WLP099 was theoretically assumed to be also the
best selected yeast strain for production of ethanol but were unable conduct proper
experiment due to practical handling error at the start of the experiment, while the WLP004
were able to produce the most biomass in this paper (Table 2), but weren’t able to produce
ethanol production as much or more than the WLP500 yeast strains. For future experiment,
these procedures were suggested to be repeated again in order to minimise the misconducted
experimental error in this paper.
Justification
The paper has been reassessed for improvement following the peer-reviews from two users
and self-improvement on scientific paper from website (11, 12). Based on the advice and
critics that the peers provided, changes has been made in the Abstract, Introduction, Results
and Discussion section. The critics from the peers are mostly on grammatical error and
construction, inconsistent of past and present tense, unclear and misunderstanding in
Introduction, Results and Discussion section, and the begin with a brief background on the
article in the Abstract section rather than to begin with aim of the article. The Abstract has
been improved by adding a general introduction and background on fermentation. The phrase
and one tense per paragraph were fixed with clarity. The introduction, results and discussion
sections has been improved. The sentence from previous introduction, “Yeast was traced
back 3,000 yeast that the Egyptians and Babylonians utilised the fermentation of wild yeast to
alveolate the bread (1) and was later then discovered by Louis Pasteur in 1845..” has been
improved to “Yeast can be traced back 3,000 years where the Egyptians and Babylonians
utilised the fermentation of wild yeast to alveolate the bread and is then later discovered by
Louis Pasteur in 1845…” and the citation and in-text citation has been rearranged and placed
at the end of each paragraph. The misunderstanding and misinterpretation in the results
section has been revised to improve the clarity and the flow for the readers. The discussion
section has been revised by removing unnecessary long words and maintaining the
information precise and succinct. Other critics from both peers such as vague title of the
paper and usage of passive voice are irrelevant for this paper for it is part of the assignment
criteria.
References
1. JoVE. 2014. An Introduction to Saccharomyces cerevisiae. The Journal of Visualized
Experiments.
2. COFALEC. 2014. Without yeast and fermentation, no bread and no beer! COFALEC
The E.U. Yeast Industry.
3. Pastor RG, Torrado RP, Garre E, Matallana E. 2011. Recent Advances in Yeast
Biomass Production. Chp 11.doi:10.5772/19458. (Article.)
4. Zaragoza S. 2014. University of Texas Austin Engineer Converts Yeast Cells into
‘Sweet Crude’ Biofuel. The Univeristy of Texas at Austin.
5. Krieger C. 2008. Saccharomyces cerevisiae Pathway: superpathway of glucose
fermentation. YeastCyc Biochemical Pathways.
6. Leskovac V, Trivić S, Pericin D. 2002. The three zinc-containing alcohol
dehydrogenases from baker’s yeast, Saccharomyces cerevisiae. FEMS Yeast
Research. 2(4): p.481-94.
7. Portela P, Moreno S. 2006. Glucose-dependent activation of protein kinase A
activity in Saccharomyces cerevisiae and phosphorylation of its TPK1 catalytic
subunit. Cellular Signalling. 18(7): p.1072-86.
8. Kellermann E, Seeboth PG, Hollenberg CP. 1986. Analysis of the primary
structure and promoter function of a pyruvate decarboxylase gene (PDC1) from
Sacchomyces cerevisiae. Nucleic Acids Research. 14(22): p.8963-77.
9. Vieira ED, Andrietta MGC, Andrietta SR. 2013. Yeast biomass production: a new
approach in glucose-limited feeding strategy. Brazilian Journal of Microbiology.
44(2): p.551-8.
10. Sprague GF, Jr. 1977. Isolation and characterization of a Saccharomyces cerevisiae
mutant deficient in pyruvate kinase activity. Journal of Bacteriology. 130(1): p.23241.
11. On-Line Resources Home. 2011. The Structure, Format, Content, and Style of a
Journal-Style Scientific Paper. p1-17.
12. Anonymous. Twenty-One Suggestions for Writing Good Scientific Paper: Notes on
Writing Papers and Theses.
<http://course1.winona.edu/mdelong/ecolab/21%20suggestions.html>