Transformation of Citric Acid to Acetic Acid,Acetoin and Diacetyl by Wine Making Lactic Acid Bacteria

A decrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC 27307. a malo-lactic bacterium, grown on the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malolactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid.

Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cell-free extract were optimal at pH 6.0. Divalent cations such as Mn²⁺, Mg²⁺, Co²⁺ and Zn²⁺ activated the citrate lyase. The citrate lyase was completely inhibited by EDTA, Hg²⁺ and Ag²⁺ . The acetoin formation from pyruvic acid was significantly stimulated by thiamine pyrophosphate and CoCl₂, and inhibited by oxaloacetic acid. Specific activities of the citrate lyase and acetoin formation were considerably variable among the six strains of malo-lactic bacteria examined. Some activities of irreversible reduction of diacetyl to acetoin were found in the cell-free extracts of four of the malo-lactic bacteria strains and the optimal pH was 6.0 for this activity of Leu. mesenteroides.     

Transformation of Vinca protoplasts mediated by Agrobacterium spheroplasts

Summary Vinca rosea protoplasts and Agrobacterium tumefaciens spheroplasts harboring octopine-type Ti plasmid were mixed and treated with polyethylene glycol or polyvinyl alcohol, which facilitated the introduction of spheroplasts into plant protoplasts. After the protoplasts had been kept at 40° C for 4 days, bacteria were found to be completely eliminated from the medium. Among treated protoplasts 1–2 per 1,000 formed colonies on the Murashige and Skoog medium (1962) lacking hormones. When the colonies were isolated and subcultured, they could be maintained as clones. Octopine, an amino acid specific to crown gall, was detected in half of these clones. The phenotypic features of these putative transformants were compared but did not show any coincidental tendencies in relation to color, hardness, form, growth rate, or octopine production. The significance of this system in transformation of higher plant cells is discussed.     

Anaerobic Phenol Degradation by Microorganisms of Swine Manure

Swine manure contains diverse groups of aerobic and anaerobic bacteria. An anaerobic bacterial consortium containing sulfate-reducing bacteria (SRB) and acetate-utilizing methanogenic bacteria was isolated from swine manure. This consortium used phenol as its sole source of carbon and converted it to methane and CO₂. The sulfate-reducing bacterial members of the consortium are the incomplete oxidizers, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. The methanogenic bacteria of the consortium converted the acetic acid to methane. When a methanogen inhibitor was used in the culture medium, phenol was converted to acetic acid by the SRB, but the acetic acid did not undergo further metabolism. On the other hand, when the growth of SRB in the consortium was suppressed with a specific SRB inhibitor, namely, molybdenum tetroxide, the phenol was not degraded. Thus, the metabolic activities of both the sulfate-reducing bacteria and the methanogenic bacteria were essential for complete degradation of phenol. Received: 31 January 1997 / Accepted: 7 March 1997     

Methanogenesis from furfural by defined mixed cultures

Methanogenesis from furfural by defined mixed cultures was studied. Under sulfate-reducing conditions, a Desulfovibrio strain was used as the furfural-degrading species producing acetic acid. This sulfate-reducing bacterium (SRB) Desulfovibrio strain B is an incomplete oxidizer, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. Introduction of acetate-utilizing methanogenic archaeon Methanosarcina barkeri 227 converted acetic acid to methane. This well-defined mixed consortium used furfural as its sole source of carbon and converted it to methane and CO₂. In the mixed culture, when a methanogen inhibitor was used in the culture medium, furfural was converted to acetic acid by the Desulfovibrio strain B, but acetic acid did not undergo further metabolism. On the other hand, when the growth of Desulfovibrio in the consortium was suppressed with a specific SRB inhibitor, namely molybdate, furfural was not degraded. Thus, the metabolic activities of both Desulfovibrio strain B and M. barkeri 227 were essential for the complete degradation of furfural. Received: 15 August 2001 / Accepted: 20 September 2001     

Carbohydrate Metabolism by the Acetic Acid Bacteria

Vitamin requirements for the growth of the acetic acid bacteria were investigated extensively on a. taxonomical viewpoint and the following new findings were pointed out. Neither Acetobacter nor Intermediate strain required vitamin for the growth.

Gluconobacter required generally pantothenic acid. And some strains belonging to it did moreover somewhat of thiamine, nicotinic acid and p-aminobenzoic acid, although there was a difference of requirements between strains even in the same species. Riboflavin, pyridoxine, vitamin B₁₂, folic acid, biotin and inositol were unnecessary for the growth of the acetic acid bacteria. A taxonomical division of the acetic acid bacteria based on the vitamin requirements agreed well with that on basis of the oxidative activities for carbohydrates.     

Cloning of a gluconate/polyol dehydrogenase gene from <Emphasis Type="Italic">Gluconobacter suboxydans</Emphasis> IFO 12528, characterisation of the enzyme and its use for the production of 5-ketogluconate in a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain

A 5-ketogluconate (5-KGA)-forming membrane quinoprotein, gluconate dehydrogenase, was isolated from Gluconobacter suboxydans strain IFO 12528 and partially sequenced. Partial sequences of five internal tryptic peptides were elucidated by mass spectrometry and used to isolate the two adjacent genes encoding the enzyme (EBI accession no. AJ577472). These genes share close homology with sorbitol dehydrogenase from another strain of G. suboxydans (IFO 3255). Substrate specificity of gluconate 5-dehydrogenase (GA 5-DH) turned out to be quite broad, covering many polyols, amino derivatives of carbohydrates, and simple secondary alcohols. There is a broad correlation between the substrate specificity of GA 5-DH and the empirical Bertrand-Hudson rule that predicts the specificity of oxidation of polyols by acetic acid bacteria. Escherichia coli transformed with the genes encoding gluconate dehydrogenase were able to convert gluconic acid into 5-KGA at 75% yield. Furthermore, it was found that 5-KGA can be converted into tartaric acid semialdehyde by a transketolase. These results provide a basis for designing a direct fermentation-based process for conversion of glucose into tartaric acid.     

Expression of membrane-bound dehydrogenases from a mother of vinegar metagenome in Gluconobacter oxydans

Acetic acid bacteria are well-known for their membrane-bound dehydrogenases rapidly oxidizing a variety of substrates in the periplasm. Since many acetic acid bacteria have not been successfully cultured in the laboratory yet, studying membrane-bound dehydrogenases directly from a metagenome of vinegar microbiota seems to be a promising way to identify novel variants of these enzymes. To this end, DNA from a mother of vinegar was isolated, sequenced, and screened for membrane-bound dehydrogenases using an in silico approach. Six metagenomic dehydrogenases were successfully expressed using an expression vector with native promoters in the acetic acid bacterium strain Gluconobacter oxydans BP.9, which is devoid of its major native membrane-bound dehydrogenases. Determining the substrates converted by these enzymes, using a whole-cell DCPIP assay, revealed one glucose dehydrogenase with an enlarged substrate spectrum additionally oxidizing aldoheptoses, D-ribose and aldotetroses, one polyol dehydrogenase with an extreme diminished spectrum but distinguishing cis and trans-1,2-cyclohexandiol and a completely new secondary alcohol dehydrogenase, which oxidizes secondary alcohols with a hydroxyl group at position 2, as long as no primary hydroxyl group is present. Three further dehydrogenases were found with substrate spectra similar to known dehydrogenases of G. oxydans 621H.

     

以pH为扰动因子探究电化学厌氧消化产甲烷代谢通量与微生物的关系

【背景】电化学厌氧消化(electrochemical anaerobic digestion,EAD)系统的代谢途径由具备不同功能的微生物所主导,其代谢通量与功能微生物丰度、活性及群落结构相关。【目的】探究EAD产甲烷代谢通量与微生物的关系。【方法】采用代谢通量分析(metabolic flux analysis,MFA)方法,以pH为扰动因子得到微生物群落与产甲烷通量的响应关系。【结果】pH 7.5扰动时产甲烷通量最大为0.398 4±0.029 3,较对照组(pH 6.9)的0.297 4±0.012 7和扰动组(pH 6.3)的0.136 5±0.012 0分别提高了25%和65%。另外,平均有33.8%±3.1%的氢气(通量)用于还原二氧化碳产甲烷和乙酸,平均有21.0%±2.6%的乙酸(通量)转化为甲烷。此外,产甲烷通量与Mariniphaga、Methanosaeta和Desulfomicrobium的丰度呈正相关,与Sedimentibacter的丰度呈负相关且影响显著。【结论】在EAD产甲烷体系中产甲烷菌和产酸菌共存时,pH值略大于7.0的环境有利于甲烷的生成,改变E...     

Influence of sulfates and operational parameters on volatile fatty acids concentration profile in acidogenic phase

Anaerobic treatment of distillery wastewaters containing high sulfate concentrations was carried out on a two-phase process. The acidogenic phase was operated so as to produce the more favourable intermediates for methanogenic bacteria coupled with maximum sulfate removal. Sulfate removal was directly affected by pH and dilution rate (D). The maximum sulfate removal and acetic acid production was achieved at pH 6.6 and D=0.035 h^–1. A linear relationship between acetic acid produced and sulfate removal was observed, indicating that acetic acid was mainly produced by sulfate reducing bacteria with important operational advantages. Higher concentrations of butyric acid were obtained at low pH values and high dilution rates.     

比较基因组学解析谷物醋醋醅中巴氏醋杆菌和欧洲驹形杆菌的功能差异

【目的】基于比较基因组分析,探究镇江香醋醋醅中不同醋酸菌的功能差异。【方法】利用分离培养技术结合16SrRNA基因全长测序获得不同分类地位的醋酸菌;应用比较基因组学结合发酵性能实现不同醋酸菌生长和代谢的差异比较。【结果】巴氏醋杆菌和欧洲驹形杆菌为镇江香醋醋醅中的主要醋酸菌。其中,欧洲驹形杆菌的GC含量更高、基因组更大。功能注释结果表明巴氏醋杆菌和欧洲驹形杆菌的碳水化合物、氨基酸相关基因数量及种类差异较大,欧洲驹形杆菌的碳水化合物活性酶数量更多。相比巴氏醋杆菌,欧洲驹形杆菌中富集的功能差异基因主要参与磷酸戊糖途径、脂肪酸生物合成、果糖和甘露糖代谢等代谢途径。验证结果表明欧洲驹形杆菌可通过产生更多的乙醇脱氢酶、乙醛脱氢酶和大量的ATP,并改变细胞膜脂肪酸组成来提高乙醇的转化率。【结论】明确了巴氏醋杆菌和欧洲驹形杆菌基因之间的差异。欧洲驹形杆菌通过更多的能量积累、更高的乙醇转化相关酶酶活力和细胞膜脂肪酸组成的改变,来改善胞内微环境以适应高酸环境。本研究得到的结果可加深对不同醋酸菌耐酸机制的理解。     

Formation and regeneration ofHalobacterium spheroplasts

Spheroplasts ofHalobacterium cutirubrum were formed upon suspension of cell pellets in 0.1M MES buffer, pH 7.0, containing 0.5M sucrose, 0.25M NaCl, and 0.01M MgCl₂. The spheroplasts regenerated into rod-shaped bacteria when plated on a complex medium containing 15% (wt/vol) sucrose, undergoing several divisions as spherical bodies before the rod shape developed. The frequency of regeneration was approximately 5% of the total spheroplasts plated. The yield of regenerants was increased significantly (to approximately 35%) when bovine serum albumin was present in the spheroplasting buffer and dilution media. The conditions for spheroplast formation and regeneration inH. cutirubrum were also found effective forHalobacterium salinarium but not forHalobacterium halobium.NRCC Paper no. 23080.     

Production of lactic acid from hemicellulose extracts by <Emphasis Type="Italic">Bacillus coagulans</Emphasis> MXL-9

Bacillus coagulans MXL-9 was found capable of growing on pre-pulping hemicellulose extracts, utilizing all of the principle monosugars found in woody biomass. This organism is a moderate thermophile isolated from compost for its pentose-utilizing capabilities. It was found to have high tolerance for inhibitors such as acetic acid and sodium, which are present in pre-pulping hemicellulose extracts. Fermentation of 20 g/l xylose in the presence of 30 g/l acetic acid required a longer lag phase but overall lactic acid yield was not diminished. Similarly, fermentation of xylose in the presence of 20 g/l sodium increased the lag time but did not affect overall product yield, though 30 g/l sodium proved completely inhibitory. Fermentation of hot water-extracted Siberian larch containing 45 g/l total monosaccharides, mainly galactose and arabinose, produced 33 g/l lactic acid in 60 h and completely consumed all sugars. Small amounts of co-products were formed, including acetic acid, formic acid, and ethanol. Hemicellulose extract formed during autohydrolysis of mixed hardwoods contained mainly xylose and was converted into lactic acid with a 94% yield. Green liquor-extracted hardwood hemicellulose containing 10 g/l acetic acid and 6 g/l sodium was also completely converted into lactic acid at a 72% yield. The Bacillus coagulans MXL-9 strain was found to be well suited to production of lactic acid from lignocellulosic biomass due to its compatibility with conditions favorable to industrial enzymes and its ability to withstand inhibitors while rapidly consuming all pentose and hexose sugars of interest at high product yields.     

Fermentative production of acetic acid from various pure and natural cellulosic materials by Clostridium lentocellum SG6

Clostridium lentocellum SG6 fermented various pure crystalline cellulosic materials efficiently with maximum acetic acid yield (gram acetic acid/gram substrate) of 0.67, at low substrate (8 g l⁻¹) concentration. The strain grew poorly on crude biopolymers but fermented them easily after alkali treatment, when grown with 8 g substrate l⁻¹ concentration of alkali-extracted cotton straw (AECS), paddy straw (AEPS) and sorghum stover (AESS) etc. The acetic acid to substrate (A/S) ratios were similar to those obtained with pure cellulosic materials. An increase in substrate concentration led to a decreased A/S ratio and a decreased percentage of substrate degraded. At high substrate concentration of 75 g filter paper l⁻¹, the strain SG6 converted 63.2 g filter paper into 31.28 g acetic acid l⁻¹. At 100 g l⁻¹ concentrations, AECS and AEPS served as the best substrates for acetic acid production when compared with other biopolymers. A maximum amount of 30.98 and 30.86 g acetic acid was produced from 70.6 g AEPS and 70.1 g AESS l⁻¹ of medium by strain SG6, respectively. Acetic acid production of 0.67 g g⁻¹ pure cellulose (Whatman No. 1 filter paper), 0.63 g g⁻¹ of alkali-treated cotton straw (AECS) are the highest among the cellulolytic bacteria reported so far in mono culture fermentations with pure and native cellulosic materials. This revised version was published online in July 2006 with corrections to the Cover Date.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

Numerical analysis of Frateur's phenotypic data on acetic acid bacteria

The phenotypic data published by J. Frateur on 35 strains of acetic acid bacteria were analyzed numerically. The four clusters obtained correspond very well to Frateur's suboxydans, mesoxydans, oxydans and peroxydans groups, on which the classification of the acetic acid bacteria in the 8th and 9th edition of Bergey's manual is based.     

Dynamics and Biodiversity of Populations of Lactic Acid Bacteria and Acetic Acid Bacteria Involved in Spontaneous Heap Fermentation of Cocoa Beans in Ghana

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as “Weissella ghanaensis,” was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named “Acetobacter senegalensis” (A. tropicalis-like) and “Acetobacter ghanaensis” (A. syzygii-like).     

Design, Development, and Use of Molecular Primers and Probes for the Detection of Gluconacetobacter Species in the Pink Sugarcane Mealybug

Molecular tools for the species-specific detection of Gluconacetobacter sacchari, Gluconacetobacter diazotrophicus, and Gluconacetobacter liquefaciens from the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae) were developed and used in polymerase chain reactions (PCR) and in fluorescence in situ hybridizations (FISH) to better understand the microbial diversity and the numerical significance of the acetic acid bacteria in the PSMB microenvironment. The presence of these species in the PSMB occurred over a wide range of sites, but not in all sites in sugarcane-growing areas of Queensland, Australia, and was variable over time. Molecular probes for use in FISH were also designed for the three acetic acid bacterial species, and shown to be specific only for the target species. Use of these probes in FISH of “squashed” whole mealybugs indicated that these acetic acid bacteria species represent only a small proportion of the microbial population of the PSMB. Despite the detection of Glac. sacchari, Glac. diazotrophicus, and Glac. liquefaciens by PCR from different mealybugs isolated at various times and from various sugarcane-growing areas in Queensland, Australia, these bacteria do not appear to be significant commensals in the PSMB environment.     

Translocation of colicin from the receptor to the inner cell membrane: function of the peptidoglycan layer

Sensitivity of spheroplasts (prepared in two ways) of a colicin-sensitive strain, of colicinresistant and of colicin-tolerant mutants and of strains immune to colicins E1 and E2 was estimated and compared. Generally, the removal of the peptidoglycan layer brought about a slight nonspecific support for colicin translocation across the cell wall in sensitive,tolB tolerant and immune bacteria.tolB spheroplasts were colicin E1-sensitive, but E2-insensitive. Spheroplasts were always fragile and lysed spontaneously, especially those produced by lysozyme. Bacteria carryingtolA, tolQ andtolR mutations kept their colicin insensitivity as spheroplasts, just as the resistant ones. Bacteria rendered colicinogenic and hence colicin-immune turned to high colicin sensitivity in spheroplast form. The results indicate a change in plasma membrane associated with the spheroplast formation.     

Diversity of the total bacterial community associated with Ghanaian and Brazilian cocoa bean fermentation samples as revealed by a 16 S rRNA gene clone library

Cocoa bean fermentation is a spontaneous process involving a succession of microbial activities, starting with yeasts, followed by lactic acid bacteria and acetic acid bacteria. So far, all microbiological studies about cocoa bean fermentation were based on culture-dependent (isolation, cultivation, and identification), or, more recently, culture-independent (PCR-DGGE, or polymerase chain reaction denaturing gradient gel electrophoresis) methods. Using a metagenomic approach, total DNA was extracted from heap and box fermentations at different time points and from different locations (Ghana and Brazil, respectively) to generate a 16 S rDNA clone library that was sequenced. The sequencing data revealed a low bacterial diversity in the fermentation samples and were in accordance with the results obtained through culture-dependent and a second, culture-independent analysis (PCR-DGGE), suggesting that almost all bacteria involved in the fermentation process are cultivable. One exception was the identification by 16 S rDNA library sequencing of Gluconacetobacter species of acetic acid bacteria that were not detected by the two other approaches. The presence of Enterobacteriaceae related to Erwinia/Pantoea/Tatumella, as revealed by 16 S rDNA library sequencing, suggests an impact of these bacteria on fermentation.     

<Emphasis Type="SmallCaps">d</Emphasis>-Hexosaminate production by oxidative fermentation

Microbial production of d-hexosaminate was examined by means of oxidative fermentation with acetic acid bacteria. In most strains of acetic acid bacteria, membrane-bound d-glucosamine dehydrogenase (synonymous with an alternative d-glucose dehydrogenase distinct from quinoprotein d-glucose dehydrogenase) oxidized d-hexosamines to the corresponding d-hexosaminates in a stoichiometric manner. Conversion of d-hexosamines to the corresponding d-hexosaminates was observed with growing cells of acetic acid bacteria, and d-hexosaminate was stably accumulated in the culture medium even though d-hexosamine was exhausted. Since the enzyme responsible is located on the outer surface of the cytoplasmic membrane, and the enzyme activity is linked to the respiratory chain of the organisms, resting cells, dried cells, and immobilized cells of acetic acid bacteria were effective catalysts for d-hexosaminate production. d-Mannosaminate and d-galactosaminate were also prepared for the first time by means of oxidative fermentation, and three different d-hexosaminates were isolated from unreacted substrate by a chromatographic separation. In this paper, d-hexosaminate production by oxidative fermentation carried out mainly with Gluconobacter frateurii IFO 3264 is exemplified as a typical example.