Zymomonas Mobilis Strain Research Articles

Open fermentative production of fuel ethanol from food waste by an acid-tolerant mutant strain of Zymomonas mobilis

The aim of present study was to develop a process for open ethanol fermentation from food waste using an acid-tolerant mutant of Zymomonas mobilis (ZMA7-2). The mutant showed strong tolerance to acid condition of food waste hydrolysate and high ethanol production performance. By optimizing fermentation parameters, ethanol fermentation with initial glucose concentration of 200g/L, pH value around 4.0, inoculum size of 10% and without nutrient addition was considered as best conditions. Moreover, the potential of bench scales fermentation and cell reusability was also examined. The fermentation in bench scales (44h) was faster than flask scale (48h), and the maximum ethanol concentration and ethanol yield (99.78g/L, 0.50g/g) higher than that of flask scale (98.31g/L, 0.49g/g). In addition, the stable cell growth and ethanol production profile in five cycles successive fermentation was observed, indicating the mutant was suitable for industrial ethanol production.

Droplet Microfluidic Technique for the Study of Fermentation

We demonstrate a technique that uses microdroplets for culturing and selecting bacterial cultures in a model biotechnological application. We propose an assay for determination of ethanol concentration that provides increased dynamic range and is compatible with droplet microfluidic screening technologies. The assay comprises two enzymes—alcohol oxidase (AOX) and horseradish peroxidase (HRP)—and a colorimetric readout system of phenol-4-sulfonic acid (PSA) and 4-aminoantipyrine (4-AAP). The microdroplet method provides high repeatability (a relative error of measured ethanol concentration < 5%), high specificity for ethanol, low consumption of reagents and wide dynamic range (1–70 g·L-1) compared to existing assays. We report the use of this method in a screen of ethanol generation efficiency of Zymomonas mobilis (strain 3881) against the concentration of glucose in the culture media.

Potential in bioethanol production from various ethanol fermenting microorganisms using rice husk as substrate

Nachaiwieng W, Lumyong S, Pratanapol R, Yoshioka K, Khanongnuch C. 2015. Potential in bioethanol production fromvarious ethanol fermenting microorganisms using rice husk as substrate. Biodiversitas 16: 320-326. Rice husk was investigated as thepotential substrate for bioethanol fermentation. It was collected from five locations in northern Thailand and found that the maincomponent of rice husk approximately 51-54% (w/w) was holocellulose. The sugar composition in rice husk holocellulose was glucose,xylose and arabinose in the ratio 66.68, 27.61 and 5.71%, respectively. Before further fermentation, acid and alkali pretreatment of ricehusk were prior investigated and 2% (w/v) NaOH at 130oC for 30 min was proved to be the most suitable pretreatment method withoutfermenting inhibitors generation. Then, rice husk hydrolysate obtained by enzymatic saccharification with Meicelase enzyme was usedas carbon sources for ethanol fermentation in comparison among 11 ethanol fermenting microorganisms including 3 strains ofSaccharomyces cerevisiae, 3 strains of Zymomonas mobilis, 3 strains of Kluyveromyces marxianus and 2 strains of pentose sugarfermenting microbes, Candida shehatae TISTR 5843 and Pichia stipitis BCC 15191. All three strains of Z. mobilis exhibited the bestethanol fermentation yield, giving the ethanol yield of 0.48 g g-1 available monosaccharides and fermentation profile of each individualgenus was also demonstrated. However, some unutilized sugars still remained in rice husk fermenting medium, therefore, conversion tovaluable products or optimization of co-culture ethanol fermentation needs to be further investigated.

Adaptive laboratory evolution of ethanologenic Zymomonas mobilis strain tolerant to furfural and acetic acid inhibitors.

Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84% theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89%. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.

Improving a recombinant Zymomonas mobilis strain 8b through continuous adaptation on dilute acid pretreated corn stover hydrolysate.

BackgroundComplete conversion of the major sugars of biomass including both the C5 and C6 sugars is critical for biofuel production processes. Several inhibitory compounds like acetate, hydroxymethylfurfural (HMF), and furfural are produced from the biomass pretreatment process leading to ‘hydrolysate toxicity,’ a major problem for microorganisms to achieve complete sugar utilization. Therefore, development of more robust microorganisms to utilize the sugars released from biomass under toxic environment is critical. In this study, we use continuous culture methodologies to evolve and adapt the ethanologenic bacterium Zymomonas mobilis to improve its ethanol productivity using corn stover hydrolysate.ResultsA turbidostat was used to adapt the Z. mobilis strain 8b in the pretreated corn stover liquor. The adaptation was initiated using pure sugar (glucose and xylose) followed by feeding neutralized liquor at different dilution rates. Once the turbidostat reached 60% liquor content, the cells began washing out and the adaptation was stopped. Several ‘sub-strains’ were isolated, and one of them, SS3 (sub-strain 3), had 59% higher xylose utilization than the parent strain 8b when evaluated on 55% neutralized PCS (pretreated corn stover) liquor. Using saccharified PCS slurry generated by enzymatic hydrolysis from 25% solids loading, SS3 generated an ethanol yield of 75.5% compared to 64% for parent strain 8b. Furthermore, the total xylose utilization was 57.7% for SS3 versus 27.4% for strain 8b. To determine the underlying genotypes in these new sub-strains, we conducted genomic resequencing and identified numerous single-nucleotide mutations (SNPs) that had arisen in SS3. We further performed quantitative reverse transcription PCR (qRT-PCR) on genes potentially affected by these SNPs and identified significant down-regulation of two genes, ZMO0153 and ZMO0776, in SS3 suggesting potential genetic mechanisms behind SS3’s improved performance.ConclusionWe have adapted/evolved Z. mobilis strain 8b for enhanced tolerance to the toxic compounds present in corn stover hydrolysates. The adapted strain SS3 has higher xylose utilization rate and produce more ethanol than the parent strain. We have identified transcriptional changes which may be responsible for these phenotypes, providing foundations for future research directions in improving Z. mobilis as biocatalysts for the production of ethanol or other fuel precursors.

Release of Cell-Free Ice Nucleators from Three Recombinant Ice+Zymomonas mobilis Strains

Background/Aims: This work is a study of the ability of three recombinant Zymomonas mobilis strains to release ice nucleators into their growth medium. Methods: The recombinant ice⁺Z. mobilis cells were tested for their ability to produce cell-free ice nucleators, under three different growth temperatures and three different glucose concentrations. Results: Cell-free ice nucleators were obtained from all the recombinant ice⁺Z. mobilis cells tested. The cell-free ice nucleation activity was not affected by the glucose concentration in the growth medium or the growth temperature. The freezing temperature threshold was below -7.6°C, demonstrating a class C nucleating structure of the ice nucleation protein. The size of the ice nucleators was less than 0.22 μm and their density was estimated as 1.024 ± 0.004 g/ml by Percoll density centrifugation. The properties of the detected ice nucleators, in addition to the absence of pyruvate decarboxylase activity in the spent medium (a cytosolic marker), support that the cell-free ice nucleation activity was due to the extracellular release of ice nucleators. Conclusion: These findings indicate that the recombinant ice⁺Z. mobilis cells could be valuable for future use as a source of active cell-free ice nucleation protein.

Genetic Engineering and Improvement of a Zymomonas mobilis for Arabinose Utilization and Its Performance on Pretreated Corn Stover Hydrolyzate

A glucose, xylose and arabinose utilizing Zymomonas mobilis strain was constructed by incorporating arabinose catabolic pathway genes, araBAD encoding L-ribulokinase, L-arabinose isomerase and L-ribulose-5-phosphate- 4-epimerase in a glucose, xylose co-fermenting host, 8b, using a transposition integration approach. Further improvement on this arabinose-capable integrant, 33C was achieved by applying a second transposition to create a genomic knockout (KO) mutant library. Using arabinose as a sole carbon source and a selection pressure, the KO library was subjected to a growth-enrichment process involving continuous sub-culturing for over 120 generations. Strain 13-1-17, isolated from such process demonstrated significant improvement in metabolizing arabinose in a dilute acid pretreated, saccharified corn stover slurry. Through Next Generation Sequencing (NGS) analysis, integration sites of the transposons were identified. Furthermore, multiple additional point mutations (SNPs: Single Nucleotide Polymorphisms) were discovered in 13-1-17, affecting genes araB and RpiB in the genome. We speculate that these mutations may have impacted the expression of the enzymes coded by these genes, ribulokinase and Ribose 5-P-isomerase, thus attributing to the improvement of the arabinose utilization.

Genetic variability analysis of Zymomonas mobilis strains from the UFPEDA microorganisms collection.

Zymomonas mobilis is a Gram-negative bacterium that has drawn attention in the bioethanol industry. Besides bioethanol, this bacterium also produces other biotechnological products such as levans, which show antitumor activity. Molecular studies involving Z. mobilis have advanced to the point that allows us to characterize interspecies genetic diversity and understand their metabolism, and these data are essential for better utilization of this species. In this study, the genetic diversity of 24 strains from the Microorganisms Collection of Departamento de Antibióticos (UFPEDA) from Universidade Federal de Pernambuco were characterized. The methods used were amplified ribosomal DNA restriction analysis and diversity analysis of the internally transcribed 16S-23S rDNA spacer region (ISR). These analyses revealed low genetic variability of the 16S rDNA gene. These data confirm that these isolates are, or are closely related to, Z. mobilis. Moreover, the analysis of the ISR confirmed the genetic variability of strains deposited in the UFPEDA collection of microorganisms and grouped these strains into ten ribotypes, which can be used in the future for breeding programs and for the preservation of biodiversity. Furthermore, this study characterized the genetic variability between the UFPEDA 205/ ZAP, UFPEDA 98/AG11, and ZAG strains, which were obtained by spheroplast fusion among them. The data also indicate that there is genetic variability among the UFPEDA 202/CP4 and UFPEDA 633/ ZM4 strains, demonstrating that these important Z. mobilis strains are distinct, as suggested in previous studies.

Molecular characterization of strains of Zymomonas mobilis by sequencing the 16S ribosomal

Background Zymomonasmobilis has attracted great interest in the scientific, industrial and biotechnological due to its high potential fermentation. From the viewpoint of taxonomic Z. mobilis is the only species of the genus Zymomonas, and is subdivided into three subspecies: Z. mobilis subsp. mobilis, Z. mobilis subsp. pomaceae and Z. mobilis susp. francensis. Differentiation between these three subspecies is based on physiological tests. These tests are time consuming and often unreliable. Therefore, molecular techniques are proposed as a quick and reliable way to characterize the genetic variability of these bacteria. This study aimed to perform molecular characterization of 6 strains of Zymomonasmobilis deposited in the Collection of Microorganisms of Department of Antibiotics, Federal University of Pernambuco (UFPEDA).

Biofilm production by Zymomonas mobilis enhances ethanol production and tolerance to toxic inhibitors from rice bran hydrolysate

Microorganisms play a significant role in bioethanol production from lignocellulosic material. A challenging problem in bioconversion of rice bran is the presence of toxic inhibitors in lignocellulosic acid hydrolysate. Various strains of Zymomonas mobilis (ZM4, TISTR 405, 548, 550 and 551) grown under biofilm or planktonic modes were used in this study to examine their potential for bioconversion of rice bran hydrolysate and ethanol production efficiencies. Z. mobilis readily formed bacterial attachment on plastic surfaces, but not on glass surfaces. Additionally, the biofilms formed on plastic surfaces steadily increased over time, while those formed on glass were speculated to cycle through accumulation and detachment phases. Microscopic analysis revealed that Z. mobilis ZM4 rapidly developed homogeneous biofilm structures within 24 hours, while other Z. mobilis strains developed heterogeneous biofilm structures. ZM4 biofilms were thicker and seemed to be more stable than other Z. mobilis strains. The percentage of live cells in biofilms was greater than that for planktonic cells (54.32 ± 7.10% vs. 28.69 ± 3.03%), suggesting that biofilms serve as a protective niche for growth of bacteria in the presence of toxic inhibitors in the rice bran hydrolysate. The metabolic activity of ZM4 grown as a biofilm was also higher than the same strain grown planktonically, as measured by ethanol production from rice bran hydrolysate (13.40 ± 2.43 g/L vs. 0.432 ± 0.29 g/L, with percent theoretical ethanol yields of 72.47 ± 6.13% and 3.71 ± 5.24% respectively). Strain TISTR 551 was also quite metabolically active, with ethanol production by biofilm and planktonically grown cells of 8.956 ± 4.06 g/L and 0.0846 ± 0.064 g/L (percent theoretical yields were 48.37 ± 16.64% and 2.046 ± 1.58%, respectively). This study illustrates the potential for enhancing ethanol production by utilizing bacterial biofilms in the bioconversion of a readily available and normally unusable low value by-product of rice farming.

Improving xylose utilization by recombinant Zymomonas mobilis strain 8b through adaptation using 2-deoxyglucose

BackgroundNumerous attempts have been made to improve xylose utilization in Z. mobilis including adaptive approaches. However, no one has yet found a way to overcome the reduced xylose utilization observed in fermentations carried out in the presence of glucose as well as the inhibitory compounds found within pretreated and saccharified biomass. Our goal was to generate Z. mobilis strains that are more robust than the wildtype strain with increased productivity in fermenting the glucose and xylose present in PCS. Through adaptation in the presence of 2-deoxyglucose, we have generated Zymomonas mobilis strain #7, which is better suited to utilizing xylose in pretreated corn stover (PCS) fermentations in the presence of both glucose and model inhibitory compounds of acetate and furfural. Strain #7 over performed the parent strain 8b both on simultaneous saccharification and fermentation (SFF) of PCS and fermentation of saccharified PCS slurry. At 65% neutralized PCS liquor level, strain #7 used 86% of the xylose present in the liquor while strain 8b was not able to ferment the liquor under similar conditions. Similarly, under SSF process conditions with 20% total solids loading of PCS, strain #7 used more than 50% of the xylose present, while strain 8b did not utilize any xylose under this condition. We have further identified genetic alterations in strain #7 in relation to the parental strain 8b that may be responsible for these phenotypic enhancements.ResultsWe performed an extended lab-directed evolution of Z. mobilis strain 8b in the presence of acetate and a non-hydrolyzable glucose analogue 2-deoxyglucose. Following the adaptation, we identified and characterized numerous candidate strains and found a dramatic increase in xylose usage not only in shake flask, but also in a controlled PCS fermentation. We re-sequenced the genomes of evolved strains to identify genetic alterations responsible for these improved phenotypes, and identified two mutations that may be key to the improved xylose usage in these strains.ConclusionWe have generated Z. mobilis strain #7, which can ferment xylose efficiently in the presence of toxins present in pretreated corn stover. Genetic alterations responsible for the improvement have been identified.

Simultaneous Saccharification of Inulin and Starch Using Commercial Glucoamylase and the Subsequent Bioconversion to High Titer Sorbitol and Gluconic Acid

A new bioprocess for production of sorbitol and gluconic acid from two low-cost feedstocks, inulin and cassava starch, using a commercially available enzyme was proposed in this study. The commercial glucoamylase GA-L NEW from Genencor was found to demonstrate a high inulinase activity for hydrolysis of inulin into fructose and glucose. The glucoamylase was used to replace the expensive and not commercially available inulinase enzyme for simultaneous saccharification of inulin and starch into high titer glucose and fructose hydrolysate. The glucose and fructose in the hydrolysate were converted into sorbitol and gluconic acid using immobilized whole cells of the recombinant Zymomonas mobilis strain. The high gluconic acid concentration of 193 g/L and sorbitol concentration of 180 g/L with the overall yield of 97.3 % were obtained in the batch operations. The present study provided a practical production method of sorbitol and gluconic acid from low cost feedstocks and enzymes.

미세조류 Microcystis aeruginosa로부터 바이오 알콜의 생산

The microalgae, Microcystis aeruginosa are able to proliferate in a wide range of freshwater ecosystem. M. aeruginosa was cultivated in 25 L and 240 L race-way reactor containing modified medium with added urea 0.2 g/L, increased Fe +2 , and decreased Ca +2 ion compared to BG11 medium. Sugar contents of M. aeruginosa grown in BG11 medium, and modified medium were 120 mg/mL and 140 mg/mL respectively. Fermentation was conducted with the extract of M. aeruginosa at 30℃ for 30 h, using Saccharomyces cerevisiae (Sc), Pichia stipitis (Ps), Zymomonas mobilis (Zm), and mixed-culture of these strains (Sc + Ps +Zm). Pichia stipitis (0.7%) was found to be more suitable for producing bioalcohol from M. aeruginosa extract than other strains of Saccharomyces cerevisiae (0.45%) and Zymomonas mobilis (0.61%), while mixed-cultured of these strains showed higest productivity by 1.75%. Biomass of M. aeruginosa contains the potency to be the most renewable resource for bioalcohol fermentation.

Consolidated Bioprocessing of Lignocellulosic Feedstocks for Ethanol Fuel Production

Ethanol fuel can be produced renewably from numerous plant and waste materials, but harnessing the energy of lignocellulosic feedstocks has been particularly challenging in the development of this alternative fuel as a substitute for petroleum-based fuels. Consolidated bioprocessing has the potential to make the conversion of biomass to fuel an economical process by combining enzyme production, polysaccharide hydrolysis, and sugar fermentation into a single unit operation. This consolidation of steps takes advantage of the synergistic nature of enzyme systems but requires the use of one or a few organisms capable of producing highly efficient cellulolytic enzymes and fermenting most of the resulting sugars to ethanol with minimal byproduct formation while tolerating high levels of ethanol. In this review, conventional ethanol production, consolidated bioprocessing, and simultaneous saccharification and fermentation are described and compared. Several wild-type and genetically engineered microorganisms, including strains of Clostridium thermocellum, Saccharomyces cerevisiae, Klebsiella oxytoca, Escherichia coli, Flammulina velutipes, and Zymomonas mobilis, among others, are highlighted for their potential in consolidated bioprocessing. This review examines the favorable and undesirable qualities of these microorganisms and their enzyme systems, process engineering considerations for particular organisms, characteristics of cellulosomes, enzyme engineering strategies, progress in commercial development, and the impact of these topics on current and future research.

Engineering efficient xylose metabolism into an acetic acid-tolerant Zymomonas mobilis strain by introducing adaptation-induced mutations

The impact of the two adaptation-induced mutations in an improved xylose-fermenting Zymomonas mobilis strain was investigated. The chromosomal mutation at the xylose reductase gene was critical to xylose metabolism by reducing xylitol formation. Together with the plasmid-borne mutation impacting xylose isomerase activity, these two mutations accounted for 80 % of the improvement achieved by adaptation. To generate a strain fermenting xylose in the presence of high acetic acid concentrations, we transferred the two mutations to an acetic acid-tolerant strain. The resulting strain fermented glucose + xylose (each at 5 % w/v) with 1 % (w/v) acetic acid at pH 5.8 to completion with an ethanol yield of 93.4 %, outperforming other reported strains. This work demonstrated the power of applying molecular understanding in strain improvement.

Respiratory Chain Analysis of Zymomonas mobilis Mutants Producing High Levels of Ethanol

We previously isolated respiratory-deficient mutant (RDM) strains of Zymomonas mobilis, which exhibited greater growth and enhanced ethanol production under aerobic conditions. These RDM strains also acquired thermotolerance. Morphologically, the cells of all RDM strains were shorter compared to the wild-type strain. We investigated the respiratory chains of these RDM strains and found that some RDM strains lost NADH dehydrogenase activity, whereas others exhibited reduced cytochrome bd-type ubiquinol oxidase or ubiquinol peroxidase activities. Complementation experiments restored the wild-type phenotype. Some RDM strains seem to have certain mutations other than the corresponding respiratory chain components. RDM strains with deficient NADH dehydrogenase activity displayed the greatest amount of aerobic growth, enhanced ethanol production, and thermotolerance. Nucleotide sequence analysis revealed that all NADH dehydrogenase-deficient strains were mutated within the ndh gene, which includes insertion, deletion, or frameshift. These results suggested that the loss of NADH dehydrogenase activity permits the acquisition of higher aerobic growth, enhanced ethanol production, and thermotolerance in this industrially important strain.

Comparison of glucose/xylose co-fermentation by recombinant Zymomonas mobilis under different genetic and environmental conditions

Three xylose-fermenting recombinant Zymomonas mobilis strains containing different Peno-talB/tktA operon terminators were engineered. Each showed similar levels of foreign protein expression and xylose fermentation performance. Strain CP4-P2-1 was further used to compare the glucose/xylose co-fermentation under various cultivation environments to improve the efficiency of the process. Optimal co-fermentation was achieved at 30-34 °C and pH 5.5 using xylose-grown preculture cells giving 20.5 g ethanol/l, ethanol productivity of 0.43 g/l h and ethanol yield of 0.44 g/g at 48 h. Adverse culture conditions mainly influenced the efficiency of xylose fermentation but not glucose fermentation. The key factors affecting co-fermentation were also explored at the molecular level. This study provides valuable insights into the effective harnessing of biomass resources.

Genome-wide transcriptomic analysis of a flocculent strain of Zymomonas mobilis

ZM401, a flocculent mutant strain of Zymomonas mobilis ZM4 was studied using genome-wide transcriptomic analysis for evidence related to phenotypic changes associated with its cell-cell attachment behaviour. Batch fermentation studies with ZM401 and its parent strain ZM4 demonstrated that similar ethanol yields and productivities could be achieved with both strains indicating the potential of the flocculent strains for cost-effective cell biomass recycling with resultant high ethanol volumetric productivities. The results showed that twofold or greater differential expression occurred for 26 genes of ZM401 when compared to those of ZM4. Among these, significant over-expression was evident for the genes ZMO1083 and ZMO1084 which are associated with bacterial cellulose synthesis, while reduced expression was found for ZMO0614, ZMO0613, and ZMO0635 which are all associated with synthesis of flagella-related proteins. Both enhanced cellulose production and reduced flagella activity are likely to facilitate more stable flocculent behaviour in ZM401. From comparative DNA sequence analysis of these 26 genes, only one single point mutation was identified. This occurred at the amino acid position A525V of ZMO1055 which encodes for diguanyl cyclase/phosphoesterase which may be related to cell motility and cellulose synthesis in Z. mobilis.

Bioethanol production in batch mode by a native strain of Zymomonas mobilis

Two wild strains of Zymomonas mobilis were isolated (named as ML1 and ML2) from sugar cane molasses obtained from different farms of Santander, Colombia. Initially, selection of the best ethanol-producer strains was carried out using ethanol production parameters obtained with a commercial strain Z. mobilis DSM 3580. Three isolated strains were cultivated in a culture medium containing yeast extract, peptone, glucose and salts, at pH 6 and 32°C with stirring rate of 65 rpm during 62 h. The best results of ethanol production were obtained with the native strain ML1, reaching a maximum ethanol concentration of 79.78 g l−1. ML1 and ML2 strains were identified as Z.mobilis, according to the morphology, biochemical tests and molecular characterization by PCR of specific DNA sequences from Z. mobilis. Subsequently, the effect of different nitrogen sources on production of ethanol was evaluated. The best results were obtained using urea at a 0.73 g/l. In this case, maximum concentration of ethanol was 83.81 g l−1, with kinetic parameters of yield of ethanol on biomass (YP/X) = 69.01(g g−1), maximum volumetric productivity of ethanol (Qpmax) = 2.28 (g l−1 h−1), specific productivity of ethanol (qP) = 3.54 (h−1) and specific growth rate (μ) = 0.12 h−1. Finally, we studied the effect of different culture conditions (pH, temperature, stirring, C/N ratio) with a Placket-Burman′s experimental design. This optimization indicated that the most significant variables were temperature and stirring. In the best culture conditions a significant increase in all variables of response was achieved, reaching a maximum ethanol concentration of 93.55 g l−1.

Respiration-deficient mutants of Zymomonas mobilis show improved growth and ethanol fermentation under aerobic and high temperature conditions

Respiration-deficient mutant (RDM) strains of Zymomonas mobilis were isolated from antibiotic-resistant mutants. These RDM strains showed various degrees of respiratory deficiency. All RDM strains exhibited much higher ethanol fermentation capacity than the wild-type strain under aerobic conditions. The strains also gained thermotolerance and exhibited greater ethanol production at high temperature (39°C), under both non-aerobic and aerobic conditions, compared with the wild-type strain. Microarray and subsequent quantitative PCR analyses suggest that enhanced gene expression involved in the metabolism of glucose to ethanol resulted in the high ethanol production of RDM strains under aerobic growth conditions. Reduction of intracellular oxidative stress may also result in improved ethanol fermentation by RDM strains at high temperatures.