Bioethanol
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Lecture 2
Energetic Biotechnology
Microorganisms for fermentation in bioethanol production:
https://www.sciencedirect.com/science/article/abs/pii/S2589014X23002141
https://amb-express.springeropen.com/articles/10.1186/s13568-021-01257-x
https://www.sciencedirect.com/science/article/abs/pii/S0960148119302125
Fermentation
Saccharomyces cerevisiae �(Baker’s or Brewer’s yeasts)
Domain: Eukarya�Kingdom: Fungi�Subkingdom: Dikarya�Phylum: Ascomycota�Subphylum: Saccharomycotina�Class: Saccharomycetes�Order: Saccharomycetales�Family: Saccharomycetaceae�Genus: Saccharomyces�Species: cerevisiae
https://microbewiki.kenyon.edu/index.php/Saccharomyces_cerevisiae
https://link.springer.com/article/10.1007/s11274-018-2518-4
Since thousands of years ago, yeasts such as S. cerevisiae have been used in alcohol production especially in the brewery and wine industries.
https://www.sciencedirect.com/science/article/pii/S2405580816302424
Fermentation
Main advantages of Saccharomyces cerevisiae
https://www.mdpi.com/2311-5637/9/8/709
S. cerevisiae is closely related to human diet and life, completely non-pathogenic, and has been confirmed as a food-grade microorganism in the long history.
S. cerevisiae is a kind of single-celled eukaryotes, which not only has the characteristics of easy culture, rapid reproduction and toilless genetic manipulation similar to prokaryotes but also has the basic molecular and cell biological characteristics of typical eukaryotes.
S. cerevisiae is relatively easy to be cultured on different types of media under laboratory conditions
The living habitat of S. cerevisiae is complex and extensive, and S. cerevisiae has developed a capacity to cope with harsh environments in long-term natural evolution and artificial application.
Mature genome manipulation and gene editing techniques for S. cerevisiae led to easy operation of building specific cell factories
Fermentation
Within all fermentative microorganisms, S. cerevisiae is regarded as an excellent industrial ethanologenic organism.
The process of bioethanol production relies on the ability of S. cerevisiae to efficiently and completely ferment sugars from feedstock biomass into ethanol.
https://biologyreader.com/production-of-bioethanol.html
The nutritional conditions required by the growth and metabolism of S. cerevisiae are relatively simple. In fermentation to produce ethanol from biomass raw materials, adding exogenous nutrients in the medium is an effective stimulus to increase the ethanol yield. Adding collagen peptide to the media significantly increased the bioethanol yield under different glucose concentrations and fermentation times compared with the non-added group.
In second-generation bioethanol production, the possible untolerance of S. cerevisiae to the inhibitors in lignocellulosic hydrolysates remains a significant challenge. Adding a mixture of pyridoxine, thiamine, and biotin to propagation media could improve cell growth and ethanol yields during fermentation.
https://www.mdpi.com/2311-5637/9/8/709
Fermentation
Major strategies for promoting bioethanol synthesis in Saccharomyces cerevisiae
https://www.mdpi.com/2311-5637/9/8/709
S. cerevisiae faces complex and varied pressures in bioethanol fermentation. Physicochemical conditions, substrate concentration, toxic effects of ethanol and other factors are essential elements affecting the final yield of bioethanol. Therefore, the screening and breeding S. cerevisiae strains with better tolerance is very important.
At present, the breeding of S. cerevisiae strains is mainly focused on improving the thermo-tolerance, glucose-tolerance, and ethanol tolerance, which is the most common type of stress faced by S. cerevisiae cells in the process of bioethanol fermentation.
To increase ethanol yield, it is a research hotspot to edit and modify the genome of S. cerevisiae. For example, disrupting the alcohol dehydrogenase (ADH2) gene via complete deletion of the gene and introducing a frameshift mutation in the ADH2 locus via CRISPR/Cas9 technology showed that the ethanol yield improved by up to 74.7% compared with the yield obtained using the native strain.
Fermentation
Problems that must be solved (for bioethanol 2nd generations):
The first generation of bioethanol is produced from food crops (corn, wheat, sweet potato, etc.) with excellent fermentation characteristics. However, food crops are extremely important resources with high production and storage costs, mainly reflected in using valuable and scarce available farmland and irrigation water. Therefore, in terms of cost, producing bioethanol from food crops is not an option based on the lowest cost and highest return.
https://www.mdpi.com/2311-5637/9/8/709
Lignocellulosic biomass represents the largest resource pool worldwide and is the raw material for producing second-generation bioethanol with low cost and wide sources. One outstanding advantage of lignocellulosic biomass is that it can be obtained for ethanol production without competing for arable land and agricultural inputs with crops for human or livestock consumption. However, despite intensive research exploring lignocellulosic ethanol, this option still accounts for <1% of global ethanol production!!!!!!!! The major drawback of these feedstocks is the recalcitrance to degradation of the lignocellulosic matrix, which is comprised of covalently and hydrogen-bonded cellulose and hemicellulose polymers that are further linked to lignin in its natural state. In addition, S. cerevisiae is difficulty utilizing β-D-xylose and α-L-arabinose, the main pentoses in hemicellulose polymers.
https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-023-02295-2
Pretreatment
Selection of new strains with useful characteristics and new technological steps are always needed to improve the bioethanol yield!
Second-generation bioethanol: new methods of
low-price and easy pretreatment steps.
https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-023-02295-2
Biological pretreatment method is preferred over other pretreatment methods due to its low energy requirement, eco-friendly process with less generation of pollutants as no chemical is required and simpler one with a broad assemblage of taxonomical microorganisms available naturally for the process.
These microorganisms are selected as bacteria, fungi or actinomycetes depending upon the substrate. Biological pretreatment involves various white, brown or red rot fungus-secreting cellulolytic enzyme that breaks the bond between cellulose and lignin, increases the accessibility of cellulose, increases porosity, and lets cellulose be available for further hydrolysis and fermentation process.
Pretreatment
Biological pretreatment of lignocellulosic biomass �
Microfungi:
Macrofungi:
Bacteria:
https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-023-02295-2/tables/4
https://www.inaturalist.org/taxa/48496-Pleurotus
https://bacdive.dsmz.de/strain/130669
Pretreatment
Biological pretreatment of lignocellulosic biomass �
Biological pretreatment assisted with bacterial treatment showed higher lignin degradation than fungal pretreatment due to its easier genetic manipulation and its tolerance towards environmental conditions.
Pretreatment
https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-023-02295-2
Bacillus firmus is considered a xylanolytic enzyme-producing bacteria used to pre-treat the biomass that has enhanced glucan recovery yield by up to 74% and increases xylan removal to 30%, this subsequently increases the availability of cellulose towards its respective enzymes.
The bacterial strain of Pandoraea sp. produces manganese peroxidase, a laccase that disintegrates the recalcitrant structure of lignocellulosic biomass along with the removal of lignin and hemicellulose from the biomass.
A bacterial strain sourced from termites’ gut is Ochrobactrum oryzae which can enhance the hydrolysis output through its degradative action. After 16 days of pretreatment, cellulosic content enhances up to 22.38%, hemicellulose to 18.64% while lignin removal reaches 44.47% from the biomass with conversion efficiency of 51.92%.
https://www.semanticscholar.org/paper/Bacillus-firmus-Bacillus-lentus%3A-a-Series-or-One-Gordon-Hyde/6856e71b20e620d458907c362581bdd7b8db8dda
https://www.sciencedirect.com/science/article/abs/pii/S0304389415001077
https://www.researchgate.net/publication/327383855_Biodegradation_of_wheat_straw_by_Ochrobactrum_oryzae_BMP03_and_Bacillus_sp_BMP01_bacteria_to_enhance_biofuel_production_by_increasing_total_reducing_sugars_yield
Video (4 min):
https://www.youtube.com/watch?v=JALTcEIZoZw
Video (4 min):
https://www.youtube.com/watch?v=aiA02GlB1Kc
Video (15 min):
https://www.youtube.com/watch?v=OpEB6hCpIGM
Problems that must be solved (for bioethanol of 3rd generations):
Pretreatment
The third generation of bioethanol production is derived from microalgal biomass. The process needs to go through several additional steps such as cell wall breaking treatment, starch extraction, hydrolysis saccharification and the final fermentation process .
https://www.mdpi.com/2311-5637/9/8/709
https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-023-02295-2
Video (6 min):
https://www.youtube.com/watch?v=d5laQZbJ2mg
�Schizosaccharomyces �pombe��
Fermentation
Domain: Eukarya�Kingdom: Fungi�Subkingdom: Dikarya�Phylum: Ascomycota�Subphylum: Taphrinomycotina �Class: Schizosaccharomycetes �Order: Schizosaccharomycetales �Family: Schizosaccharomycetaceae �Genus: Schizosaccharomyces �Species: pombe
https://microbewiki.kenyon.edu/
index.php/Schizosaccharomyces_pombe
https://microbenotes.com/candida-tropicalis/
� Candida
tropicalis��
Domain: Eukarya�Kingdom: Fungi�Subkingdom: Dikarya�Phylum: Ascomycota�Subphylum: Saccharomycotina�Class: Saccharomycetes�Order: Saccharomycetales�Family: Saccharomycetaceae�Genus: Candida�Species: tropicalis
�
Scheffersomyces
stipitis
��
Fermentation
Domain: Eukarya�Kingdom: Fungi�Subkingdom: Dikarya�Phylum: Ascomycota�Subphylum: Taphrinomycotina �Class: Schizosaccharomycetes �Order: Schizosaccharomycetales �Family: Debaryomycetaceae �Genus: Scheffersomyces�Species: stipitis
https://microbewiki.kenyon.edu/index.php/Pichia_stipitis
Schematic illustration of the three-step procedures performed to obtain a high cell density of Scheffersomyces stipitis in cellular recycle batch fermentations using two temperature strategies and a short-term adaptation
Pachysolen sp. ��
Fermentation
Domain: Eukarya�Kingdom: Fungi�Subkingdom: Dikarya�Phylum: Ascomycota�Subphylum: Saccharomycotina�Class: Saccharomycetes�Order: Saccharomycetales�Family: Saccharomycetaceae�Genus: Pachysolen�Species: tannophilus
Co-cultivation of S. cerevisiae and Pachysolen tannophilus resulted in higher ethanol production
Kluyveromyces sp. ��
Domain: Eukarya�Kingdom: Fungi�Subkingdom: Dikarya�Phylum: Ascomycota�Subphylum: Saccharomycotina�Class: Saccharomycetes�Order: Saccharomycetales�Family: Saccharomycetaceae�Genus: Kluyveromyces �Species: marxianus
Sugar cane bagasse
Acid Hydrolysis
Cellulose +
Hemicellulose +
Lignin
Immobilization of yeasts on PVA (polyvinyl alcohol)
Bioethanol
A total of 18.01 g/L ethanol was produced after 10 h of cultivation with immobilized culture which was higher than that from the freely suspended culture (15.31 g/L at 12 h), resulting in a 15% increase in yield. Furthermore, the immobilized system was able to maintain constant ethanol production even after 21 days of storage, and could be reused in fermentation for at least 8 cycles. The findings showed that the nanothreads is an ideal system for repeated production while upcycling sugarcane biomass waste.
Nanothreads
Fermentation
Kluyveromyces
sp. ��
Fermentation
Lactobacillus sp.�(lactic acid bacteria)
Fermentation
Domain: Bacteria�Phylum: Bacillota�Class: Bacilli�Order: Lactobacillales�Family: Lactobacillaceae�Genus: Lactobacillus �Species: casei
https://microbewiki.kenyon.edu/index.php/Lactobacillus_acidophilus
Engineered L. casei E1 has the abilities to metabolize sucrose, glucose, and fructose of sugarcane molasses into ethanol. The ethanol yield could reach about 13.77 g/L, and carbohydrate utilization percentage is about 78.60%.
https://amb-express.springeropen.com/articles/10.1186/s13568-021-01257-x
Clostridium sp.�
Domain: Bacteria�Phylum: Bacillota�Class: Clostridia�Order: Eubacteriales�Family: Clostridiaceae�Genus: Clostridium�Species: butyricum
https://microbewiki.kenyon.edu/index.php/Clostridium
https://www.sciencedirect.com/science/article/abs/pii/S0960148120307977
Syngas can be obtained from the gasification of lignocellulosic biomass, by which most of carbon content of the biomass was converted into CO and CO2. These gases could be further utilized by carbon-fixing microorganism such as Clostridium sp. to produce ethanol as the end product.
https://www.matec-conferences.org/articles/matecconf/abs/2018/15/matecconf_rsce2018_03025/matecconf_rsce2018_03025.html#:~:text=Syngas%20can%20be%20obtained%20from,ethanol%20as%20the%20end%20product.
Syngas and charcoal were used as a substrate of Clostridium butyricum.
Fermentation
Zymomonas
mobilis �
Domain: Bacteria�Phylum: Pseudomonadota �Class: Alphaproteobacteria�Order: Sphingomonadales�Family: Zymomonadaceae�Genus: Zymomonas�Species: mobilis
Fermentation
https://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0718-07642011000600002
https://link.springer.com/chapter/10.1007/978-3-031-01241-9_18
Extractive fermentation, or liquid-liquid extraction-fermentation hybrid, is the process in which in-situ extraction is conducted to remove the product ethanol and other inhibitory compounds, thus eliminating inhibitions caused by ethanol and other inhibitors and hence increasing the ethanol yield.
An extractive fermentation system was developed to reduce the inhibitory effects of ethanol on Z. mobilis. The most biocompatible extractive solvent was shown to be iso-octadecanol.
https://www.sciencedirect.com/science/article/abs/pii/S096030851630181X
Video (6 min):
https://www.youtube.com/watch?v=lqgBukMYaA8&ab_channel=AusAgave
End of Lecture 2
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