Sustainable Production of Bioethanol by Zymomonas mobilis and Saccharomyces cerevisiae using Rice Husk and Groundnut Shell as Substrates

Background of Study: Plant waste such as rice husk and groundnut shell are generated in large amounts, these waste presents a tremendous pollution to the environment. Worldwide, these wastes are often simply dumped into landfills and oceans or used as animal feeds. The recovery of food processing wastes as renewable energy sources represents a sustainable option for the substitution of fossil energy in order to minimize environmental damages and to meet energy demands of the growing population. Aim: To produce bioethanol from rice husk and groundnut shell using local strains of Zymomonas mobilis and Saccharomyces cerevisiae. Place and Duration of Study: Conducted at the Microbiology Laboratory of Abubakar Tafawa Balewa University Bauchi, Bauchi state, Nigeria, between April to June, 2021. Methods: Groundnut shell and Rice husk were collected from local milling center. The wastes were powdered, sieved and used as carbon source. Proximate composition of the subsrate was done and the total carbohydrate was determined by difference. The sum of the percentage moisture, ash, crude lipid, crude protein and crude fibre was subtracted from 100. Zymomonas mobilis and Saccharomyces cerevisiae were isolated from rotten sweet oranges and locally fermented beverage (‘kunun-zaki’) respectively by growing them on Malt Yeast Peptone Glucose Agar (MYPGA) after which they were further screened for their ability to tolerate ethanol and they serve as organisms for fermentation. The enzyme α- amylase was used for hydrolysis. The fermented substrates were distilled at 78oC and the distillate was collected as bioethanol in a conical flask. UV-VIS spectrophotometer was used to determine the absorbance of each concentration (0, 0.2, 0.4, 0.6 and 0.8cm3) of reducing sugar content of the hydrolysates and the bioethanol produced by developing a standard curve at a wavelength of 491nm and 588nm respectively. The concentration of reducing sugar and bioethanol was determined using a reference line from the Standard curve. Results: Proximate analysis done shows that rice husk have 70.09% carbohydrates while groundnut shell has 65.09% carbohydrates. Groundnut shell yielded the highest reducing sugar of 5.096%. Rice husk yielded the lowest quantity of reducing sugar with a total yield of 2.962%. Maximum concentration of bioethanol of 0.971% was produced from the combination of Saccharomyces cerevisiae and Zymomonas mobilis from groundnut shell. The lowest concentration of 0.121% of bioethanol was produced when Saccharomyces cerevisiae was used on rice husk hydrolysates. The synergistic relationship of Saccharomyces cerevisiae and Zymomonas mobilis yielded the maximum bioethanol when compared with the yield obtained when the organisms were used singly. Zymomonas mobilis produced highest bioethanol content when the organisms are used single. Conclusion: This study demonstrates the potentiality of local strains of Saccharomyces cerevisiae and Zymomonas mobilis isolated from rotten sweet orange and locally fermented beverage (‘kunun-zaki’) to produce bioethanol by fermenting the rice husk and groundnut shell hydrolysates.

Revitalizing the ethanologenic bacterium Zymomonas mobilis for sugar reduction in high-sugar-content fruits and commercial products

The excessive consumption of sugars can cause health issues. Different strategies have been developed to reduce sugars in the diets. However, sugars in fruits and commercial products may be difficult to reduce, limiting their usage among certain populations of people. Zymomonas mobilis is a generally recognized as safe (GRAS) probiotic bacterium with the capability to produce levan-type prebiotics, and thrives in high-sugar environments with unique characteristics to be developed for lignocellulosic biofuel and biochemical production. In this study, the sugar reduction capabilities of Z. mobilis ZM4 were examined using two fruits of pear and persimmon and three high-sugar-content commercial products of two pear pastes (PPs) and one Chinese traditional wine (CTW). Our results demonstrated that Z. mobilis ZM4 can utilize sugars in fruits with about 20 g/L ethanol and less than 5 g/L sorbitol produced within 22 h using pears, and about 45 g/L ethanol and 30 g/L sorbitol produced within 34 h using persimmons. When PPs made from pears were used, Z. mobilis can utilize nearly all glucose (ca. 60 g/L) and most fructose (110 g/L) within 100 h with 40 ~ 60 g/L ethanol and more than 20 g/L sorbitol produced resulting in a final sorbitol concentration above 80 g/L. In the high-sugar-content alcoholic Chinese traditional wine, which contains mostly glucose and ethanol, Z. mobilis can reduce nearly all sugars with about 30 g/L ethanol produced, resulting in a final ethanol above 90 g/L. The ethanol yield and percentage yield of Z. mobilis in 50 ~ 60% CTW were 0.44 ~ 0.50 g/g and 86 ~ 97%, respectively, which are close to its theoretical yields—especially in 60% CTW. Although the ethanol yield and percentage yield in PPs were lower than those in CTW, they were similar to those in fruits of pears and persimmons with an ethanol yield around 0.30 ~ 0.37 g/g and ethanol percentage yield around 60 ~ 72%, which could be due to the formation of sorbitol and/or levan in the presence of both glucose and fructose. Our study also compared the fermentation performance of the classical ethanologenic yeast Saccharomyces cerevisiae BY4743 to Z. mobilis, with results suggesting that Z. mobilis ZM4 had better performance than that of yeast S. cerevisiae BY4743 given a higher sugar conversion rate and ethanol yield for sugar reduction. This work thus laid a foundation for utilizing the advantages of Z. mobilis in the food industry to reduce sugar concentrations or potentially produce alcoholic prebiotic beverages. Graphical Abstract

Zymomonas mobilis as an emerging biotechnological chassis for the production of industrially relevant compounds

Zymomonas mobilis is a well-recognized ethanologenic bacterium with outstanding characteristics which make it a promising platform for the biotechnological production of relevant building blocks and fine chemicals compounds. In the last years, research has been focused on the physiological, genetic, and metabolic engineering strategies aiming at expanding Z. mobilis ability to metabolize lignocellulosic substrates toward biofuel production. With the expansion of the Z. mobilis molecular and computational modeling toolbox, the potential of this bacterium as a cell factory has been thoroughly explored. The number of genomic, transcriptomic, proteomic, and fluxomic data that is becoming available for this bacterium has increased. For this reason, in the forthcoming years, systems biology is expected to continue driving the improvement of Z. mobilis for current and emergent biotechnological applications. While the existing molecular toolbox allowed the creation of stable Z. mobilis strains with improved traits for pinpointed biotechnological applications, the development of new and more flexible tools is crucial to boost the engineering capabilities of this bacterium. Novel genetic toolkits based on the CRISPR-Cas9 system and recombineering have been recently used for the metabolic engineering of Z. mobilis. However, they are mostly at the proof-of-concept stage and need to be further improved. Graphical Abstract

Cellulosic ethanol production by consortia of Scheffersomyces stipitis and engineered Zymomonas mobilis

Background As one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. However, the simultaneous transformation of glucose and xylose to ethanol is one of the key technologies for attaining cost-efficient lignocellulosic ethanol production at an industrial scale. Genetic modification of strains and constructing consortia were two approaches to resolve this issue. Compared with strain improvement, the synergistic interaction of consortia in metabolic pathways should be more useful than using each one separately. Results In this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in artificially simulated 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various corn stover hydrolysates with different sugar concentrations (glucose and xylose 60, 90, 120 g/L), were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56–4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12–102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Furthermore, qRT-PCR analysis of xylose/glucose transporter and other genes responsible for CCR explained the reason why the initial ratio inoculation of 1:3 in artificially simulated 80G40XRM had the best fermentation performance in the consortium. Conclusions The fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. This result showed that this strategy has potential for future lignocellulosic ethanol production.

Enhancing Secretion of Endoglucanase in Zymomonas mobilis by Disturbing Peptidoglycan Synthesis

Zymomonas mobilis ( Z. mobilis ) is a potential candidate for consolidated bioprocessing (CBP) strain in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion affected by cell wall peptidoglycan is also not well understood. Here we constructed several Penicillin Binding Proteins (PBPs)-deficient strains derivated from Z. mobilis S192 to perturb the cell wall peptidoglycan network and investigated the effects of peptidoglycan on the endoglucanase secretion. Results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBPs mutant strains, notably, △1089/0959 (4.09-fold) and △0959 (5.76-fold) in comparison to parent strains. Besides, for PBPs-deficient strains, the growth performance was not significantly inhibited but with enhanced antibiotic sensitivity and reduced inhibitor tolerance, otherwise, cell morphology was altered obviously. The concentration of intracellular soluble peptidoglycan was increased, especially for single gene deletion. Outer membrane permeability of PBPs-deficient strains was also improved, notably, △1089/0959 (1.14-fold) and △0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our finding indicated that PBPs-deficient Z. mobilis is capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness, and also acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacterias. Herein, we perturb the peptidoglycan synthesis network via knocking out PBPs ( ZMO0197 , ZMO0959 , ZMO1089 ) in order to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study can not only lay the foundation for understanding the regulatory network of cell wall synthesis but also provide guidance for the construction of CBP strains in Z. mobilis .

Metabolic Remodeling during Nitrogen Fixation in Zymomonas mobilis

Biofuels and bioproducts have the potential to serve as environmentally sustainable replacements for petroleum-derived fuels and commodity molecules. Advanced fuels such as higher alcohols and isoprenoids are more suitable gasoline replacements than bioethanol.

Functional Gene Identification and Corresponding Tolerant Mechanism of High Furfural-Tolerant Zymomonas mobilis Strain F211

Furfural is a major inhibitor in lignocellulose hydrolysate for Zymomonas mobilis. A mutant F211 strain with high furfural tolerance was obtained from our previous study. Thus, its key tolerance mechanism was studied in the present study. The function of mutated genes in F211 was identified by functional complementation experiments, revealing that the improved furfural tolerance was resulted from the C493T mutation of the ZCP4_0270 gene promoting cell flocculation and the mutation (G1075A)/downregulation of ZCP4_0970. Comparative transcriptome analysis revealed 139 differentially expressed genes between F211 and the control, CP4, in response to furfural stress. In addition, the reliability of the RNA-Seq data was also confirmed. The potential tolerance mechanism was further demonstrated by functional identification of tolerance genes as follows: (I) some upregulated or downregulated genes increase the levels of NAD(P)H, which is involved in the reduction of furfural to less toxic furfuryl alcohol, thus accelerating the detoxification of furfural; (II) the mutated ZCP4_0270 and upregulated cellulose synthetase gene (ZCP4_0241 and ZCP4_0242) increased flocculation to resist furfural stress; (III) upregulated molecular chaperone genes promote protein synthesis and repair stress-damaged proteins; and (IV) transporter genes ZCP4_1623–1,625 and ZCP4_1702–1703 were downregulated, saving energy for cell growth. The furfural-tolerant mechanism and corresponding functional genes were revealed, which provides a theoretical basis for developing robust chassis strains for synthetic biology efforts.

Zymomonas mobilis ZM4 utilizes an acetaldehyde dehydrogenase to produce acetate

Zymomonas mobilis is a promising bacterial host for biofuel production but further improvement has been hindered because some aspects of its metabolism remain poorly understood. For example, one of the main byproducts generated by Z. mobilis is acetate but the pathway for acetate production is unknown. Acetaldehyde oxidation has been proposed as the major source of acetate and an acetaldehyde dehydrogenase was previously isolated from Z. mobilis via activity guided fractionation, but the corresponding gene has never been identified. We determined that the locus ZMO1754 (also known as ZMO_RS07890) encodes an NADP+-dependent acetaldehyde dehydrogenase that is responsible for acetate production by Z. mobilis. Deletion of this gene from the chromosome resulted in a growth defect in oxic conditions, suggesting that acetaldehyde detoxification is an important role of acetaldehyde dehydrogenase. The deletion strain also exhibited a near complete abolition of acetate production, both in typical laboratory conditions and during lignocellulosic hydrolysate fermentation. Our results show that ZMO1754 encodes the major acetaldehyde dehydrogenase in Z. mobilis and we therefore rename the gene aldB based on functional similarity to the Escherichia coli acetaldehyde dehydrogenase.