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.

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.

Nutritional requirements of Zymomonas mobilis CCT 4494 for levan production

The aim of this work was to study the nutritional requirements of Zymomonas mobilis CCT 4494 for levan production in three chemically defined media. During the optimization of the fermentative process for the production of the exopolysaccharide, different concentrations of glucose, fructose and sucrose as carbon source and yeast extract as vitamin source were tested. Variations of incubation temperature and initial pH of the medium were observed. The results showed that medium containing 20.0 percent sucrose and 0.5 percent yeast extract, with initial pH of 7.0, incubated at 30°C gave a 43.0 percent yield of the biopolymer.

Influence of high osmotic pressure on sorbitol production by Zymomonas mobilis

The objective of the present work was to study the variation on the sorbitol production in relation to the concentration of sugars, (metabolizable or not) and the cultivation time. A full factorial design was used considering the factors such as sucrose and maltose concentration and cultivation time. The addition of sugars caused increases on the sorbitol production up to the concentration of 300g/L however, decreases on the sorbitol production were observed when the concentration reached values above this. Increasing the time of fermentation was statistically significant to sorbitol production, however, little increase the production was noticed after 36h.

Zymomonas mobilis produz o poliálcool sorbitol como principal subproduto. Sua formação é atribuída principalmente a sua função A produção de sorbitol foi avaliada através de um planejamento fatorial completo utilizando as variáveis concentração de sacarose, concentração de maltose e tempo de cultivo. A adição de açúcares causou um aumento na produção de sorbitol até a concentração de 300g/L, porém decréscimos na produção de sorbitol foram observados a concentrações superiores a esta. Aumento no tempo de fermentação foi estatisticamente significativo para aumentos da produção de sorbitol, porém pequeno aumento foi observado de 12 para 36 horas de cultivo.

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.

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).

Research on Exogenous Gene mRNA Levels in Zymomonas mobilis by Semi-quantitative RT-PCR / 微生物学通报

An effective RT-PCR method was developed to detect exogenous gene xylB transcript levels in Zymomonas mobilis CP4. Total RNAs without genomic DNA contamination were purified from wild-type and gene engineering strains, and were quantified to the same concentration. Then, cDNAs synthesis and PCR analysis of these samples were conducted by reverse transcription PCR. The optimal number of cycles was determined by observing amplification profile of target gene xylB and internal control gene 16s rRNA, and relative expression levels of xylB in various samples were analyzed by RT-PCR. The results indicated that the xylB transcript was not be detected in CP4, however that could be found in recombinant strains, in which xylB transcription abundance was similar. The enzyme assay furthermore confirmed that effective ex-pression of the target gene. The method provided a useful and rapid tool for detecting transcript levels of target genes from various samples of Z. mobilis.

Fermentation pattern of Zymomonas mobilis strains on different substrates—a comparative study.

The optimum conditions (pH and initial sugar concentration) of fermentation for the production of ethanol by 4 strains of Zymomonas mobilis (ATCC 10988, ATCC 12526, NRRL Β 4286 and IFO 13756) were studied. An initial sugar concentration of 15 % (w/v) at pH 7·0 was found to be optimal for the first two strains and 20 % (w/v) initial sugar at pH 7·0 was found to be optimal for the last two strains. The fermentation pattern of these strains on synthetic medium, cane juice and molasses were compared. Strain NRRL Β 4286 showed maximum ethanol production on synthetic medium while on cane juice ATCC 10988 and ATCC 12526 performed well. However, all the strains fermented molasses poorly.