RecET recombination system driving chromosomal target gene replacement in Zymomonas mobilis

Background: Zymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments. Results: The plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4. Conclusion: This approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.

Optimization of asparaginase production from Zymomonas mobilis by continuous fermentation / Otimização da produção de asparaginase de Zymomonas mobilis por fermentação contínua

A asparaginase é uma enzima usada em tratamento clínico como agente quimioterapêutico e em tecnologia de alimentos na prevenção de formação de acrilamida em alimentos fritos e assados. Asparaginase é industrialmente produzida por micro-organismos, principalmente bactérias gram negativas. Zymomonas mobilis é uma bactéria gram negativa que utiliza glicose, frutose e sacarose como fonte de carbono e é conhecida por sua eficiência para produzir etanol, sorbitol, levana, ácido glicônico, e mais recentemente, tem despertado interesse no uso desse micro-organismo na produção de asparaginase. Este trabalho teve como objetivo otimizar a produção de asparaginase de Z. mobilis por fermentação contínua, pelo uso do delineamento experimental e da metodologia da superfície de resposta, testando as variáveis: sacarose, extrato de levedura e asparagina. A condição ótima alcançada, com produção de 117,45 UI L-1 foi na taxa de diluição 0,20 h-1, utilizando 0,5 g L-1 de extrato de levedura, 20 g L-1 de sacarose e 1,3 g L-1 de asparagina. Observou-se que a relação carbono:nitrogênio (1:0,025) exerceu forte influência na resposta da atividade de asparaginase. A utilização de Z. mobilis por fermentação contínua demonstrou ser uma alternativa promissora na produção biotecnológica da asparaginase.

Asparaginase is an enzyme used in clinical treatments as a chemotherapeutic agent and in food technology to prevent acrylamide formation in fried and baked foods. Asparaginase is industrially produced by microorganisms, mainly gram-negative bacteria. Zymomonas mobilis is a Gram-negative bacterium that utilizes glucose, fructose and sucrose as carbon source and has been known for its efficiency in producing ethanol, sorbitol, levan, gluconic acid and has recently aroused interest for asparaginase production. Current assay optimizes the production of Z. mobilis asparaginase by continuous fermentation using response surface experimental design and methodology. The studied variables comprised sucrose, yeast extract and asparagine. Optimized condition obtained 117.45 IU L-1 with dilution rate 0.20 h-1, yeast extract 0.5 g L-1, sucrose 20 g L-1 and asparagine 1.3 g L-1. Moreover, carbon:nitrogen ratio (1:0.025) strongly affected the response of asparaginase activity. The use of Z. mobilis by continuous fermentation has proved to be a promising alternative for the biotechnological production of asparaginase.

Three new shuttle vectors for heterologous expression in Zymomonas mobilis

Background: Zymomonas mobilis, as a novel platform for bio-ethanol production, has been attracted more attention and it is very important to construct vectors for the efficient expression of foreign genes in this bacterium. Results: Three shuttle vectors ( pSUZM 1, pSUZM2 and pSUZM3 ) were first constructed with the origins of replication from the chromosome and two native plasmids (pZZM401 and pZZM402) of Z. mobilis ZM4, respectively. The three shuttle vectors were stable in Z. mobilis ZM4 and have 3,32 and 27 copies, respectively. The promoter Ppdc (a), from the pyruvate decarboxylase gene, was clonedinto the shuttle vectors, generatingthe expressionvectors pSUZM1(2, 3)a. The codon-optimized glucoamylase gene from Aspergillus awamori combined with the signal peptide sequence from the alkaline phosphatase gene of Z. mobilis was cloned into pSUZM1(2, 3)a, resulting in the plasmids pSUZM1a-GA, pSUZM2a-GA and pSUZM3a-GA, respectively. After transforming these plasmids into Z. mobilis ZM4, the host was endowed with glucoamylase activity for starch hydrolysis. Both pSUZM2a-GA and pSUZM3a-GA were more efficientatproducingglucoamylase thanpSUZM1a-GA. Conclusions: These results indicated that these expression vectors are useful tools for gene expression in Z. mobilis and this could provide a solid foundation for further studies of heterologous gene expression in Z. mobilis.

Fermentation of rice bran hydrolysate to ethanol using Zymomonas mobilis biofilm immobilization on DEAE-cellulose

Background The major challenges associated with the fermentation of lignocellulosic hydrolysates are the reduction in the operating cost and minimizing the complexity of the process. Zymomonas mobilis biofilm has been emerged to resolve these complexities. Biofilm has been reported to tolerate to the toxic inhibitors and easily manipulated toward the cell recycle through the cell immobilization. Results Z. mobilis ZM4 and TISTR 551 were able to develop biofilms on DEAE cellulose under the differences in the morphologies. Z. mobilis ZM4 developed homogeneous biofilm that brought DEAE fiber to be crosslinking, while Z. mobilis TISTR 551 developed heterogeneous biofilm in which crosslinking was not observed. Ethanol production under batch and repeated batch fermentation of rice bran hydrolysate containing toxic inhibitors were compared between these two biofilms. TISTR 551 biofilm produced the maximum yield (Y P/S) of 0.43 ± 0.09 g ethanol/g glucose (83.89% theoretical yield). However the repeated batch could not be proceeded due to the bacterial detachment. Z. mobilis ZM4 biofilm produced the maximum yield (Y P/S) of 0.177 ± 0.05 g ethanol/g glucose (34.74% theoretical yield) in the batch culture and the biofilm remained intact to proceed along the repeated batch. The highest ethanol yield (Y P/S) in the repeated batch of Z. mobilis ZM4 was 0.354 ± 0.07 g ethanol/g glucose (69.51% theoretical yield). Conclusions Homogeneous biofilm structure of Z. mobilis provided more recycle beneficial over the heterogeneous biofilm structure for the ethanol production from lignocellulosic hydrolysate.

Production of coenzyme Q10 by metabolically engineered Escherichia coli / 生物工程学报

Coenzyme Q10 (CoQ10) is a lipophilic antioxidant that improves human immunity, delays senility and enhances the vitality of the human body and has wide applications in pharmaceutical and cosmetic industries. Microbial fermentation is a sustainable way to produce CoQ10, and attracts increased interest. In this work, the native CoQ8 synthetic pathway of Escherichia coli was replaced by the CoQ10 synthetic pathway through integrating decaprenyl diphosphate synthase gene (dps) from Rhodobacter sphaeroides into chromosome of E. coli ATCC 8739, followed by deletion of the native octaprenyl diphosphate synthase gene (ispB). The resulting strain GD-14 produced 0.68 mg/L CoQ10 with a yield of 0.54 mg/g DCW. Modulation of dxs and idi genes of the MEP pathway and ubiCA genes in combination led to 2.46-fold increase of CoQ10 production (from 0.54 to 1.87 mg/g DCW). Recruiting glucose facilitator protein of Zymomonas mobilis to replace the native phosphoenolpyruvate: carbohydrate phosphotransferase systems (PTS) further led to a 16% increase of CoQ10 yield. Finally, fed-batch fermentation of the best strain GD-51 was performed, which produced 433 mg/L CoQ10 with a yield of 11.7 mg/g DCW. To the best of our knowledge, this was the highest CoQ10 titer and yield obtained for engineered E. coli.

Transformation of phosphotransferase system in Escherichia coli / 生物工程学报

We constructed several recombinant Escherichia coli strains to transform phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS system) and compared the characteristics of growth and metabolism of the mutants. We knocked-out the key genes ptsI and ptsG in PTS system by using Red homologous recombination in E. coli and meanwhile we also knocked-in the glucose facilitator gene glf from Zymomonas mobilis in the E. coli chromosome. Recombinant E. coli strains were constructed and the effects of cell growth, glucose consumption and acetic acid accumulation were also evaluated in all recombinant strains. The deletion of gene ptsG and ptsI inactivated some PTS system functions and inhibited the growth ability of the cell. Expressing the gene glf can help recombinant E. coli strains re-absorb the glucose through Glf-Glk (glucose facilitator-glucokinase) pathway as it can use ATP to phosphorylate glucose and transport into cell. This pathway can improve the availability of glucose and also reduce the accumulation of acetic acid; it can also broaden the carbon flux in the metabolism pathway.

Recombinant expression, purification and characterization of a novel DyP-type peroxidase in Escherichia coli / 生物工程学报

Dye-decolorizing peroxidase (DyP-type peroxidase) represents a group of heme-containing peroxidases able to decolour various organic dyes, most of which are xenobiotics. To identify and characterize a new DyP-type peroxidase (ZmDyP) from Zymomonas mobilis ZM4 (ATCC 31821), ZmDyP was amplified from the genomic DNA of Z. mobilis by PCR, and cloned into the Escherichia coli expression vector pET-21b(+). Alignment of the amino acid sequence of ZmDyP with other members of the DyP-type peroxidases revealed the presence of the active site conserved residues D149, R239, T254, F256 as well as the typical GXXDG motif, indicating that ZmDyP is a new member of the Dyp-type peroxidase family. pET-21b(+) containing ZmDyP gene was expressed in E. coli by IPTG induction. The expressed enzyme was purified by Ni-Chelating chromatography. SDS-PAGE analysis of the purified enzyme revealed a molecular weight of 36 kDa, whereas activity staining gave a molecular weight of 108 kDa, suggesting that the enzyme could be a trimer. In addition, ZmDyP is a heme-containing enzyme as shown by a typical heme absorption peak of Soret band. Moreover, ZmDyP showed high catalytic efficiency with 2, 2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) as a substrate. These results enrich the pool of DyP-type peroxidases and lay a foundation for further studies.

Ethanol fermentation of sugarcane molasses by Zymomonas mobilis MTCC 92 immobilized in Luffa cylindrica L. sponge discs and Ca-alginate matrices

Bio-ethanol production from cane molasses (diluted to 15 % sugar w/v) was studied using the bacterium, Zymomonas mobilis MTCC 92 entrapped in luffa (Luffa cylindrica L.) sponge discs and Ca-alginate gel beads as the immobilizing matrices. At the end of 96 h fermentation, the final ethanol concentrations were 58.7 ± 0.09 and 59.1 ± 0.08 g/l molasses with luffa and Ca-alginate entrapped Z. mobilis cells, respectively exhibiting 83.25 ± 0.03 and 84.6 ± 0.02 % sugar conversion. There was no statistical significant difference (Fischer's LSD) in sugar utilization (t = 0.254, p <0.801) and ethanol production (t =-0.663, p <0.513) between the two immobilization matrices used. Further, the immobilized cells in both the matrices were physiologically active for three more cycles of operation with less than 15 % decrease in ethanol yield in the 4th cycle, which was due to some leakage of cells. In conclusion, luffa sponge was found to be equally good as Ca-alginate as a carrier material for bacterial (Z. mobilis. cell immobilization for ethanol production. Further, it has added advantages such as it is cheap, non-corrosive and has no environmental hazard.

Evaluation on glucose-xylose co-fermentation by a recombinant Zymomonas mobilis strain / 生物工程学报

Co-fermentation of glucose and xylose is critical for cellulosic ethanol, as xylose is the second most abundant sugar in lignocellulosic hydrolysate. In this study, a xylose-utilizing recombinant Zymomonas mobilis TSH01 was constructed by gene cloning, and ethanol fermentation of the recombinant was evaluated under batch fermentation conditions with a fermentation time of 72 h. When the medium containing 8% glucose or xylose, was tested, all glucose and 98.9% xylose were consumed, with 87.8% and 78.3% ethanol yield, respectively. Furthermore, the medium containing glucose and xylose, each at a concentration of 8%, was tested, and 98.5% and 97.4% of glucose and xylose was fermented, with an ethanol yield of 94.9%. As for the hydrolysate of corn stover containing 3.2% glucose and 3.5% xylose, all glucose and 92.3% xylose were consumed, with an ethanol yield of 91.5%. In addition, monopotassium phosphate can facilitate the consumption of xylose and enhance ethanol yield.

Evaluation of supplementation of sucrose medium on the synthesis of Zymomonas mobilis bio-products / Avaliação da suplementação do meio de sacarose na formação de bio–produtos por Zymomonas mobilis

The effect of the variables pantothenic acid, yeast extract and sodium chloride, as well as the cell permeabilization technique, were investigated on the formation of levan, ethanol, sorbitol and biomass of Zymomonas mobilis, using a 24-1 fraction factorial design. Cell growth was determined by turbidimetry at 605 nm, relating it to a biomass with a dry weight calibration curve. Reducing sugars were quantified according to Somogyi and Nelson. Total sugars were quantified by the phenol-sulfuric acid method, sorbitol by HPLC and ethanol. The levan produced was precipitated by the addition of absolute ethanol and quantified in fructose units. In levan biosynthesis, the variable that had the largest contribution was cell condition. The results suggested that the factors that most affected biomass and ethanol formation were sodium chloride concentration and cell condition that affected negatively on production. For sorbitol, the variable that had a significant effect was permeabilization, which decreased its synthesis. Studies to amplify the range of established factors would be important.

A influência das variáveis: ácido pantotênico, extrato de levedura, cloreto de sódio, e a técnica de permeabilização celular foram investigadas na formação de levana, sorbitol, etanol e biomassa de Zymomonas mobilis utilizando um delineamento estatístico fatorial fracionado 24-1. A biomassa foi determinada por turbidimetria, Os açúcares redutores foram quantificados por Somogy e Nelson, açúcar total por Fenol Sulfúrico, sorbitol por HPLC e etanol por micro-destilação. A levana produzida foi precipitada com etanol absoluto e determinada como unidade de frutose. Na biossíntese de levana, a variável que mais contribuiu foi a condição celular. Os resultados sugerem que, para a formação da biomassa e etanol, os fatores que mais interferiram foram a concentração de cloreto de sódio e a condição celular que influencia negativamente a produção. Para o sorbitol, a variável que teve efeito significativo foi a permeabilização celular que atuou diminuindo a sua síntese. Estudos que ampliam a faixa de variação dos fatores estabelecidos são interessantes.

Production of ethanol from mesquite (Prosopis juliflora (SW) D. C) pods mash by Zymomonas mobilis and Saccharomyces cerevisiae

This study aimed to assess the use of mesquite pods hydrated mash as biomass for the growth of Saccharomyces cerevisiae UFEPEDA-1012 and Zymomonas mobilis UFEPEDA-205 and for ethanol production using a submerged fermentation. A 2³ factorial design was used to analyze the effects of the type of microorganism, time of fermentation and condition of cultivation on the ethanol production in mesquite pods mash (30 g 100 mL-1). From the obtained results the hydrated mesquite pods mash presented as a good substrate for the growth of S. cerevisiae and Z. mobilis in comparison to the standard media. The effect that most affected the ethanol production was the type of microorganism. The highest ethanol concentration (141.1 gL-1) was found when Z. mobilis was cultivated in mesquite pods mash under static condition for 36 hrs. Ethanol production by S. cerevisiae was higher (44.32 gL-1) after 18 hrs of fermentation under static condition. According to these results, the mesquite pods could be known as an alternative substrate to be used for biotechnological purposes, mainly for ethanol production.

Efecto de adición de iones hierro y zinc sobre la producción de etanol de dos cepas recombinantes de Saccharomyces cerevisiae / The effect of adding iron and zinc ions to ethanol production from two recombinant Saccharomyces cerevisiae strains

La producción de etanol por fermentación es influenciada por la presencia de iones metálicos como hierro y zinc dado que son cofactores de la enzima alcohol deshidrogenasa. El estudio de este efecto permitiría identificar el comportamiento de los microorganismos fermentadores en sustratos industriales que contienen altas concentraciones de este tipo de iones. Este trabajo evaluó la producción de biomasa, los azúcares residuales y la producción de etanol por fermentación de tres cepas de S. cerevisiae, CBS8066, recombinantes GG570-CIBI y GG570-CIBII, bajo el efecto de la adición de hierro a 0, 50 y 150 M, y zinc a 0 y 50 M. Las cepas presentaron inhibición en la producción de biomasa y etanol bajo efecto de iones de hierro y zinc, siendo dicha inhibición mayor al estar en presencia de zinc o alta concentración de hierro. GG570-CIBI mostró disminución en producción de biomasa de 4 g/L y una caída en producción de etanol de 40% en el tratamiento 150 M hierro-50 M zinc (con respecto al tratamiento basal). GG570-CIBII fue la menos afectada con inhibición en la producción de etanol inferior a 11% a las 20 h de fermentación. Adicionalmente, presentó la mayor producción de etanol cuando hubo adición de 150 M Fe con o sin adición de zinc, siendo dicha producción entre un 9 y 14% superior a la de las cepas CBS8066 y GG570-CIBI respectivamente, bajo las mismas condiciones. Posteriormente, GG570-CIBII será evaluada en sustratos industriales debido a su menor inhibición en la producción de etanol, permitiendo así obtener mejores rendimientos.

The ethanol production by fermentation is influenced by the presence of metallic ions like iron and zinc because these are alcohol dehydrogenase enzyme cofactors. The study of this effect would allow for identifying the behavior of microorganisms in industrial substrates that contain high concentrations of this kind of ions. This work evaluated biomass production, residual sugars and ethanol production by fermentation of three S. cerevisiae strains, CBS8066, recombinants GG570-CIBI and GG570-CIBII, under the effect of the addition of ferrous ion at 0, 50 and 150 M and zinc ion at 0 and 50 M. The strains showed inhibition on biomass and ethanol production under the effect of zinc and ferrous ions, however, this inhibition was greater in the presence of zinc or iron at high concentration. GG570-CIBI showed reduction in biomass production of 4 g/L and an ethanol production drop of 40 % in the treatment 150 M iron–50 M zinc (with respect to the basal treatment). GG570-CIBII was the less affected with an inhibition on ethanol production below 11 % at 20 h of fermentation. Additionally, GG570-CIBII presented the greatest ethanol production when 150 M iron was added to the culture medium with or without zinc addition. In this case, the production was 9 and 14 % greater than ethanol production of CBS8066 and GG570-CIBI respectively, at the same conditions. Later, GG570-CIBII will be evaluated in industrial substrates due to its lower ethanol production inhibition, allowing for obtaining better yields.

In vivo assessment of possible probiotic properties of Zymomonas mobilis in a Wistar rat model

In recent years the incorporation of probiotic bacteria into foods has received increasing scientific interest for health promotion and disease prevention. The safety and probiotic properties of Zymomonas mobilis CP4 (UFPEDA-202) was studied in a Wistar rat model fed the 10(9) colony forming units (cfu)/mL-1 of the assayed strain for 30 days. No abnormal clinical signs were noted in the group receiving viable cells of Z. mobilis and water (control) during the period of the experiment. There were no significant difference (p > 0.05) in feed intake and weight gain among mice fed the Z. mobilis in comparison to the control group. No bacteria were found in blood, liver and spleen of any animals. Mice receiving Z. mobilis showed significantly differences (p < 0.05) in total and differential leucocytes count, excepting for neutrophils, after the experimental period. Otherwise, it was not found in control group. Histological examination showed that feeding mice with Z. mobilis caused no signs of adverse effects on gut, liver and spleen. From these results, Z. mobilis CP4 (UFEPEDA-202) is likely to be nonpathogenic and safe for consumption, and could have a slight modulating effect on immunological performance in mice.

El pulque: características microbiológicas y contenido alcohólico mediante espectroscopia Raman / Pulque: Microbiological characteristics and alcohol content by Raman spectroscopy

A partir de cuatro muestras de pulque (bebida mexicana prehispánica) en diferentes etapas de fermentación denominadas aguamiel, semilla, contrapunta y corrida; se aislaron e identificaron un hongo levaduriforme, un cocobacilo Gram negativo y un bacilo Gram positivo pertenecientes a los géneros Saccharomyces sp, Zymomonas sp, y Lactobacillus sp. El consorcio desarrollado fue el responsable de la fermentación, alcohólica, ácida y viscosa propias de esta bebida tradicional Mexicana. El contenido alcohólico varió en función del tiempo de fermentación alcanzando porcentajes de 10.35 (v/v) y 9.01 (v/v) determinados por cromatografía de gases y espectroscopia Raman para la etapa denominada ®corrida¼. El producto terminado tiene un elevado contenido de proteínas (1 g/L), azúcares reductores (4.75 g/L) y poblaciones microbianas en ordenes de 45x108 UFC/mL para Saccharomyces sp, 41x107 UFC/mL para Zymomonas sp y 34x106 UFC/mL para Lactobacillus sp, que le dan a la bebida un elevado valor nutricional y los microorganismos aislados podrían tener un efecto benéfico sobre sistema digestivo al ser consumido por vía oral.

Uso de matérias primas da agroindústria: garapa e extrato de levedura: na produção de asparaginase por Zymomonas mobilis CP4 / Utilization of raw materials from agroindustry: sugar cane juice and yeast extract: for asparaginase production by Zymomonas mobilis CP4

Garapa e extrato de levedura foram usados na produção de asparaginase por Zymomonas mobilis CP4. Na otimização utilizou metodologia de superfície de resposta com 2 variáveis (extrato de levedura e asparagina) em 3 níveis (1,0; 5,5 e 10,0 g/L) e uma repetição do ponto central. A fermentação em batelada utilizou garapa diluída a 8 % (P/V) de Açúcares Totais e inóculo de Zymomonas mobilis CP4 na concentração de 2 mg/mL. Após a fermentação de 18 horas, a maior produção obtida de asparaginase foi de 9,75 U/L em extrato de levedura em 5,5 g/L e asparagina em 1,0 g/L.

Sugar cane juice and yeast extract have been used for asparaginase production by Z. mobilis CP4. A complete factorial design of two variables (yeast extract and asparagin) at three levels (1.0; 5.5 and 10.0 g/L) with one replication at the central point was used. Batch fermentation utilised sugar cane juice diluted at 8 % (W/V) of Total Sugars and an inoculum of 2 mg of cells/mL. After fermentation time of 18 hours, the highest production of asparaginase was 9.75 U/L using both yeast extract (5.5 g/L) and asparagin (1.0 g/l).

Application of fractional factorial design to levan production by Zymomonas mobilis

Levan is a non-toxic, biologically active, extra cellular polysaccharide composed solely by fructose units. Optimization of levan production by Zymomonas mobilis strain ZAG-12 employing a 2(4-1) fractional factorial design was performed to analyze the influence of the temperature (20, 25 e 30°C) agitation (50, 75 e 100 rpm), and the initial concentrations of both sucrose (150, 200 e 250 g.L-1) and yeast extract (2.0, 3.5 e 5.0g.L-1) on final levan concentration. Aerobic fermentation was performed batchwise in 500mL Pyrex flasks for 72 hours. Biomass, ethanol, levan and sucrose were determined at beginning and also at end of the fermentations. The experiments showed that the final levan concentration depended on initial sucrose concentration, temperature and agitation velocity and that the initial concentration of yeast extract did not influence levan production. However, when the production of ethanol and biomass were considered, it became evident that yeast extract was a significant variable. The best conditions for levan production occurred at 100 rpm agitation, 20°C and 250g.L-1 of initial sucrose resulting in 14.67g.L-1 of levan.

Levana é um polissacarídeo extracelular, biologicamente ativo, não tóxico, contendo em sua estrutura apenas frutose. A maximização da produção de levana, por via fermentativa, pela linhagem de Zymomonas mobilis ZAG-12, foi estudada utilizando-se um planejamento fatorial de dois níveis 2(4-1), variando-se as concentrações iniciais de sacarose (150, 200 e 250 g.L-1) , extrato de levedura (2.0, 3.5 e 5.0 g.L-1), temperatura (20, 25 e 30°C) e agitação (50, 75 e 100 rpm). As fermentações foram desenvolvidas por processos descontínuos em frascos Pyrex roscados, de 500 mL, contendo 300 mL de meio a base de sacarose, por 72 horas. No início e ao final do processo, foram dosados: biomassa, etanol, levana e sacarose como açúcares redutores totais. A análise dos dados mostra que o aumento da produção de levana depende tanto dos efeitos da concentração inicial de sacarose, temperatura e agitação, isoladamente, quanto da interação entre agitação e temperatura na faixa experimental estudada. O extrato de levedura não afeta a produção de levana, entretanto, quando a resposta é produção de etanol e biomassa, fica evidente que essa variável é significativa. Os resultados demonstraram que as melhores condições para a produção em batelada ocorreram com 250g/L de sacarose inicial, 100 rpm de agitação, a 20°C.

Synthesis of sorbitol by Zymomonas mobilis under high osmotic pressure

The bacterium Zymomonas mobilis presents potential for sorbitol production when grown in culture medium with high sugar concentration. Sorbitol is produced and accumulated in the periplasma of the bacterium to protect the cells from the harmful effects of high osmotic pressure that results from the action of invertase on sucrose. The conversion of sucrose into glucose and fructose increases the osmolarity of the medium. However, an excessive increase in the osmotic pressure may decrease the sorbitol production. In this work Saccharomyces cerevisiae invertase was added two media containing sucrose 200 and 300 g.L-1. Sorbitol production in sucrose at 200 g.L-1 was 42.35 and 38.42 g.L-1, with and without the invertase treatment, respectively. In the culture medium with 300 g.L-1 sucrose, production reached 60.4 g.L-1 and with invertase treatment was 19.14 g.L-1. These results indicated that the excessive rise in osmotic pressure led to a significant decrease in sorbitol production by the Zymomonas mobilis bacterium in the sucrose medium treated with invertase.

A bactéria Zymomonas mobilis, apresenta potencial para produção de sorbitol quando crescida em meio com alta concentração de açúcar. O sorbitol produzido é acumulado no periplasma da bactéria para conter os efeitos prejudiciais da elevada pressão osmótica, que resulta pela ação da enzima invertase, que promove hidrólise da sacarose. A conversão da sacarose em glicose e frutose aumentando a osmolaridade do meio. Entretanto, um aumento excessivo na pressão osmótica pode inibir a produção de sorbitol pela bactéria. Este trabalho empregou invertase de Saccharomyces cerevisiae nos meios de fermentação com sacarose a 200 e 300 g.L-1. A produção de sorbitol no meio com sacarose a 200 g.L-1 foi de 42,35 g.L-1 e 38,42 g.L-1 com e sem tratamento com invertase respectivamente. No meio com 300 g.L-1 sem tratamento, a produção foi de 60,42 e com tratamento 19,14 g.L-1. Estes resultados indicaram que a elevação excessiva da pressão osmótica, pela adição de invertase levou a uma diminuição significativa na produção de sorbitol pela bactéria Zymomonas mobilis.

Ethanolic fermentation of sucrose, sugarcane juice and molasses by Escherichia coli strain ko11 and Klebsiella oxytoca strain P2

As bactérias recombinantes Escherichia coli KO11 e Klebsiella oxytoca P2 fermentaram sacarose a etanol. Em meio mínimo com 2% ou 12% de sacarose, KO11 apresentou, respectivamente, 75% e 41% do rendimento máximo teórico (0,54g de etanol/g de sacarose). No caldo Luria-Bertani (LB) com até 8% de sacarose, KO11 apresentou rendimento de aproximadamente 94-96% e com 12% de sacarose, KO11 apresentou cerca de 69% de rendimento (44,5g de etanol/L). A porcentagem do rendimento máximo teórico obtida com P2 em meio mínimo com 2% de sacarose foi de 55% e com 12% de sacarose foi de 47%. Em LB com 2 ou 4% de sacarose, P2 apresentou 94-95% do rendimento máximo teórico, porém somente cerca de 73% com 8% de sacarose (31,4g de etanol/L) e 58% com 12% de sacarose (37,5 g/L). A produtividade volumétrica em LB contendo 2% de sacarose foi de 0,41 g/L/h para KO11 e de 1,1 g/L/h para P2, enquanto que em LB com 12% de sacarose, a produtividade foi 0,96 g/L/h (KO11) e 1,4 g/L/h (P2). Durante a fermentação do caldo de cana, E. coli KO11 e K. oxytoca P2 produziram, respectivamente, 39,4 g de etanol/L e 42,1 g/L quando suplementado com 0,5% de extrato de levedura, micronutrientes e tiamina. No caldo de cana suplementado com os reagentes do meio LB, KO11 apresentou forte inibição da fermentação alcoólica, produzindo apenas 23,0 g de etanol/L, enquanto que P2 produziu 44,2 g/L. A produção de etanol por KO11 e P2, no caldo de cana suplementado com a) 0,2% de sulfato de amônio foi, respectivamente: 25,3 e 30,2 g/L, b) com sulfato de amônio e micronutrientes: 24,9 e 31,6 g/L, c) com sulfato de amônio, micronutrientes e tiamina: 25,6 e 37,5 g/L. Durante a fermentação do melaço, E. coli KO11 apresentou baixa produção de etanol e alta produção de ácido láctico. K. oxytoca P2 produziu 25 g de etanol/L a partir de melaço diluído 10X em água, com ou sem adição de 0,5% de extrato de levedura e 27,8 g/L com reagentes do caldo LB após 96h. P2 produziu 24,5, 26,9, e 28,0 g de etanol/L em melaço diluído ...

The effect of Zymomonas mobilis culture on experimental Schistosoma mansoni infection

Camundongos C57Bl/10 do sexo masculino, infectados com Schistosoma mansoni foram distribuídos nos grupos misto, profilático e curativo. Cultura de Zymomonas mobilis foi administrada oralmente aos camundongos. Uma proteção de 61% foi observada no grupo curativo (p<0,05). Os estudos histopatológicos dos fígados e intestinos mostraram resultados similares.

Five Computer Simulation Studies of Whole-Genome Fragment Assembly: The Case of Assembling Zymomonas mobilis ZM4 Sequences

An approach for genome analysis based on assembly of fragments of DNA from the whole genome can be applied to obtain the complete nucleotide sequence of the genome of Zymomonas mobilis. However, the problem of fragment assembly raise thorny computational issues. Computer simulation studies of sequence assembly usually show some abnormal assemblage of artificial sequences containing repetitive or duplicated regions, and suggest methods to correct those abnormalities. In this paper, we describe five simulation studies which had been performed previous to the actual genome assembly process of Zymomonas mobilis ZM4.