Aerobic submerged fermentation by acetic acid bacteria for vinegar production: Process and biotechnological aspects

Strictly aerobic acetic acid bacteria (AAB) have a long history of use in fermentation processes, and the conversion of ethanol to acetic acid for the production of vinegar is the most well-known application.At the industrial scale, vinegar is mainly produced by submerged fermentation, which refers to an aerobic process in which the ethanol in beverages such as spirits, wine or cider is oxidized to acetic acid by AAB. Submerged fermentation requires robust AAB strains that are able to oxidize ethanol under selective conditions to produce high-titer acetic acid. Currently submerged fermentation is conducted by unselected AAB cultures, which are derived from previous acetification stocks and maintained by repeated cultivation cycles.In this work, submerged fermentation for vinegar production is discussed with regard to advances in process optimization and parameters (oxygen availability, acetic acid content and temperature) that influence AAB activity. Furthermore, the potential impact arising from the use of selected AAB is described.Overcoming the acetification constraints is a main goal in order to facilitate innovation in submerged fermentation and to create new industry-challenging perspectives.     

Microbiological Studies of Coli-aerogenes Bacteria

During investigations on the metabolisms of glucose by coli-aerogenes bacteria, it was found that the bacteria accumulated a large amount of α-ketoglutaric acid under aerobic conditions such as shaking culture, while lactic acid was ascertained to be produced anaerobically by the bacteria as was already known.     

耐乙酸乳杆菌的筛选及其产乳酸特性

【背景】耐受乙酸的乳酸菌是传统谷物醋醋酸发酵过程中产生乳酸及其风味衍生物的重要功能微生物。【目的】从镇江香醋醋醅中分离鉴定具有耐乙酸特性的乳酸菌,并评价不同条件下该菌株的产乳酸能力。【方法】利用4%(体积比)乙酸含量的MRS培养基分离耐乙酸乳酸菌;对其进行16S rRNA基因鉴定、基因组测序、形态观察以及生理生化特性研究;考察不同乙酸浓度、葡萄糖浓度、发酵温度和时间对菌株产乳酸能力的影响。【结果】分离得到一株可耐受6%乙酸的乳杆菌Lactobacillus sp. JN500903;在厌氧静置、接种量5%、乙酸浓度5%、葡萄糖浓度40 g/L、发酵温度37°C、发酵时间10 d条件下,该菌株乳酸产量为16.1 g/L。【结论】乳杆菌JN500903能够耐受6%乙酸浓度,具有在酸性环境下合成乳酸的能力,有一定的应用潜力。     

醋酸菌耐酸机理及其群体感应研究新进展

醋酸菌(acetic acid bacteria,AAB)是一类严格好氧的革兰氏阴性细菌,因其乙醇氧化生成醋酸能力强、高耐醋酸等特性而成为食醋发酵的主要工业菌种。醋酸菌的耐酸性对于高酸度食醋生产具有重要意义。随着醋酸菌的蛋白组学及基因组学研究的深入,其糖代谢、蛋白质代谢、脂代谢及应激响应等分子机制或过程也得到更多的阐释;葡糖醋杆菌中有关群体感应系统的研究报道则为从信号通路角度探索醋酸菌的耐酸机制提供了新的思路,进而对于高耐酸醋酸菌的选育以及醋酸发酵工艺的优化具重要的参考意义。本文在简介蛋白组、基因组研究的基础上,着重综述醋酸菌群体感应的研究进展。     

Wine vinegar production using a noncommercial 100-litre bubble column reactor equipped with a novel type of dynamic sparger

This paper describes batch and semicontinuous acetic acid fermentations for wine vinegar production carried out with Acetobacter pasteurianus, and an industrial strain using a noncommercial 100-L bubble column reactor equipped with a novel type of gas-liquid dynamic sparger. Results showed acetification rates with this fermentor (i.e., an overall acetic acid productivity of 1. 8 g/L/h and yield of 94%) similar to that of the Frings acetator and higher as compared to others fermentors in current industrial use in Spanish wine vinegar factories, and a linear relationship between overall productivity and kLa with different operating conditions and fermentation scales. Copyright 1999 John Wiley & Sons, Inc.     

Factors Affecting Organic Acid Production by Sourdough (San Francisco) Bacteria

Previous workers from this laboratory observed considerable variation in the proportions of acetic and lactic acids produced in pure broth culture as compared to consistently high proportions of acetic acid produced in the sourdough and flour suspension systems. In the latter the proportion of acetic acid was always in the range of 20 to 35% of the total, whereas in pure broth culture frequently less than 5% acetic acid was produced. In the natural environment, the sourdough bacteria, tentatively identified as lactobacilli, coexist with a yeast, Saccharomyces exiguus, and this study was undertaken to determine whether this yeast or flour ingredients including glucose or other factors were involved in this variable production of acetic acid. The proportion of acetic acid produced in broth culture on maltose, the preferred carbohydrate source, was found to depend almost entirely on the degree of aeration. Essentially anaerobic conditions, as obtained by thorough evacuation and flushing with CO(2) or N(2), resulted in very low (5% or less) proportions of acetic acid. Aerobic conditions, achieved by continuous shaking in cotton-plugged flasks, yielded high levels (23 to 39% of the total) of acetic acid. Similar effects of aeration were observed with glucose as the substrate, although growth was considerably slower, or in nonsterile flour suspension systems. It is theorized that, under aerobic conditions, the reduced pyridine nucleotides generated in the dissimilation of carbohydrate are oxidized directly by molecular oxygen, thereby becoming unavailable for the reduction of the acetyl phosphate intermediate to ethyl alcohol, the usual product of anaerobic dissimilation of glucose by heterofermentative lactic acid bacteria. Comparative studies with known strains of homo- and heterofermentative lactobacilli showed similar effects of aeration only on the heterofermentative strains, lending additional support to the tentative grouping by previous workers from this laboratory of the sourdough bacteria with the heterofermentative lactobacilli.     

Isolation and Identification of Acetic Acid Bacteria for Submerged Acetic Acid Fermentation at High Temperature

Industrial vinegar production by submerged acetic acid fermentation has been carried out using Acetobacter strains at about 30°C. To obtain strains suitable for acetic acid fermentation at higher temperature, about 1,100 strains of acetic acid bacteria were isolated from vinegar mash, soils in vinegar factories and fruits, and their activities to oxidize ethanol at high temperature were examined. One of these strains, No. 1023, identified as Acetobacter aceti, retained full activity to produce acetic acid in continuous submerged culture at 35°C and produced 45% of activity at 38°C, while the usual strain of A. aceti completely lost its activity at 35°C. Thus the use of this strain may reduce the cooling costs of industrial vinegar production.     

Aerobiosis–anaerobiosis transition has a significant impact on organic acid production by Corynebacterium glutamicum

Corynebacterium glutamicum is known to produce organic acids under anaerobic culture conditions, in particular, lactic, succinic, and acetic acids. Our study is focused on acetic and succinic acid production using a lactate dehydrogenase-deficient strain of C. glutamicum. Usually, with this bacterium, the organic acid production process is based on an initial aerobic growth phase, followed by a rapid deoxygenation and an anaerobic production phase. In our study, we demonstrated that this strategy was unfavorable for the production of organic acids. Conversely, we showed that applying the best transition strategy based on progressive deoxygenation significantly increased the concentration of organic acids up to 640%. This was observed either by applying controlled dissolved oxygen concentrations or by decreasing the steps of gas flow rates. Our results also showed that applying constant oxygen transfer flux throughout the culture, and thus in the absence of the anaerobic phase, promoted constant production yields (approximately 0.5 mol of succinate or acetate per mole of glucose). In this case, acetate production (120 mM) was favored over succinate production (132 mM), resulting in a decrease in the molar ratio of products (succinate/acetate) from 4.8 to 1.1 between progressive deoxygenation and constant OTR cultures.     

Process-engineering characterization of small-scale bubble columns for microbial process development

Volumetric oxygen transfer rates and power inputs were estimated by a model of the formation of primary gas bubbles at the static sparger (sinter plate) of small-scale bubble columns and a common mass-transfer correlation for bubbles rising in a non-coalescent Newtonian electrolyte solution of low viscosity. Estimations were used to assess the dimensioning and possibilities of small-scale bubble column application with an height/diameter ratio of about 1. Estimations of volumetric oxygen transfer rates (<0.16 s^-1) and power inputs (<100 W m^-3) with a mean pore diameter of the static sparger of 13 µm were confirmed as function of the superficial air velocity (<0.6 cm s^-1) by measurements using an Escherichia coli fermentation medium. Small-scale bubble columns are thus to be classified between shaking flasks and stirred-tank reactors with respect to the oxygen transfer rate, but the maximum volumetric power input is more than one magnitude below the power input in shaking flasks, which is of the same order of magnitude as in stirred-tank reactors. A small-scale bubble columns system was developed for microbial process development, which is characterized by handling in analogy to shaking flasks, high oxygen transfer rates and simultaneous operation of up to 16 small-scale reactors with individual gas supply in an incubation chamber.     

Growth of silicone-immobilized bacteria on polycarbonate membrane filters, a technique to study microcolony formation under anaerobic conditions.

A technique was developed to study microcolony formation by silicone-immobilized bacteria on polycarbonate membrane filters under anaerobic conditions. A sudden shift to anaerobiosis was obtained by submerging the filters in medium which was depleted for oxygen by a pure culture of bacteria. The technique was used to demonstrate that preinduction of nitrate reductase under low-oxygen conditions was necessary for nonfermenting, nitrate-respiring bacteria, e.g., Pseudomonas spp., to cope with a sudden lack of oxygen. In contrast, nitrate-respiring, fermenting bacteria, e.g., Bacillus and Escherichia spp., formed microcolonies under anaerobic conditions with or without the presence of nitrate and irrespective of aerobic or anaerobic preculture conditions.     

Effects of the oxygen transfer rate on ferrous iron oxidation by Thiobacillus ferrooxidans

Ferrous iron oxidation by Thiobacillus ferrooxidans was studied in shake flasks and a bubble column under different aeration conditions. The maximum biooxidation rate constant was affected by oxygen transfer only at low aeration intensities. At oxygen transfer rates higher than 0.03 mmol O₂ l⁻¹ min⁻¹, the maximum biooxidation rate constant was about 0.050 h⁻¹ in both shake flasks of different size and the bubble column. The oxygen transfer rate could be used as a basis for scaling up bioreactors for ferrous iron biooxidation by T. ferrooxidans.     

用生料制备功能性双歧醋生产工艺的实验室研究

目的将双歧杆菌、醋酸菌、酵母菌和粉碎的制醋原料及麸曲共同发酵,通过生料制醋的方法来制备功能性双歧醋。方法将粉碎的玉米与麸曲、酵母液、麸皮和水搅拌均匀,使其经过液态糖化和酒精发酵后,接入醋酸菌和双歧杆菌(二者比例为1∶1),同时加入辅料,进行醋酸发酵,当检测到醋酸酸度为5.0%~7.5%时,加入食盐终止发酵,经过过滤,除菌澄清得到功能性双歧醋。结果双歧醋的最终醋酸度为3.2%,外观红棕色,光泽度好,清澈透明,无沉淀和悬浮物。总菌数:醋酸菌为3.3×1011/m l,双歧杆菌为1.9×107/m l;活菌数:醋酸菌为1.7×1011/m l,双歧杆菌为6.8×106/m l;大肠菌群数3个/100 m l;致病菌:不得检出。结论双歧杆菌及其代谢物可以在双歧醋中存活,生料固态发酵制备双歧醋的方法可行。     

一株番茄表面着生醋酸菌的分离鉴定及产酸条件优化

醋酸菌是食醋酿造过程中的关键菌种,性能优良的菌种对于产品品质的提升意义重大。以分离自番茄表面的产醋酸菌为研究对象,通过生理生化指标鉴定、16S rRNA编码基因比对及系统发育树构建等方法对其种类进行鉴定,并通过单因素实验、正交实验对鉴定为醋酸菌的菌株进行培养条件优化。结果表明,所分离的3株醋酸生产菌中,BQ-1被鉴定为醋酸杆菌属(Acetobacteraceae),在以酵母粉为主要氮源,蔗糖为主要碳源的培养基中,其最高产酸量为1823 g·L-1。由于该菌株在番茄表面具有很强的生长能力,因此有望应用于番茄果醋的酿造。     

Examination of reproducibility in microbiological degradation experiments

Experimental data indicate that certain microbiological degradation experiments have a limited reproducibility. Nine identical batch experiments were carried out on 3 different days to examine reproducibility. A pure culture, isolated from soil, grew with toluene as the only carbon and energy source. Toluene was degraded under aerobic conditions at a constant temperature of 28 °C. The experiments were modelled by a Monod model – extended to meet the air/liquid system, and the parameter values were estimated using a statistical nonlinear estimation procedure. Model reduction analysis resulted in a simpler model without the biomass decay term. In order to test for model reduction and reproducibility of parameter estimates, a likelihood ratio test was employed. The limited reproducibility for these experiments implied that all 9 batch experiments could not be described by the same set of parameter values. However, experiments carried out the same day (within the same run) were more uniform than experiments carried out on different days (between runs), and a common set of parameter estimates could be accepted for experiments within runs, but not for experiments from different runs. The limited reproducibility may be caused by variability in the preculture, or more precisely, variations in the physiological state of the bacteria in the precultures just before used as inoculum.     

Performance of a cell recycle bubble column fermentor with a membrane filter module for continuous vinegar production

The steady-state performance of a bubble column combined with a membrane filter module for cell separation and recycle is investigated numerically in the case of vinegar fermentation. The one-dimensional dispersion model for describing the longitudinal mixing of the liquid phase is employed. Kinetic expressions and their parameter values are taken from the available literature. Several characteristics of this fermentor system namely the concentration profiles of cells, substrate and product, the viability of viable cells relative to total cells, the washout condition for cells and the productivity of acetic acid are discussed. The average cell viability in the whole column and the critical dilution rate for washout are presented as equations. Low levels of the axial mixing are found to enhance the vinegar productivity. The optimum dilution rate giving the maximum productivity is determined and both are shown as figures with the Peclet number, the recycle ratio and the bleed ratio as parameters.     

Cultivation of recombinant E. coli and production of fusion protein in 60-1 bubble column and airlift tower loop reactors.

E. coli K12 with multicopy plasmid (lambda PR-promoter and temperature-sensitive lambda cI 857 repressor) was cultivated in 60-l bubble column and airlift tower loop reactors. The medium composition, cell concentration, and intracellulary enzyme activity were monitored on-line during batch, fed-batch, and continuous cultivations. The specific growth rates, cell mass yield coefficients, plasmid stabilities, productivities of the amount of active fusion protein (beta-galactosidase activity), concentrations and yields of acetic acid, and volumetric oxygen transfer coefficient were evaluated for different medium compositions and cultivation conditions. The enzyme activity was also monitored during the temperature induction. The results evaluated in the 60-l bubble column and airlift tower loop reactors are compared with those evaluated in a 1-1 stirred-tank reactor.     

Characterization of acetic acid bacteria in traditional acetic acid fermentation of rice vinegar (komesu) and unpolished rice vinegar (kurosu) produced in Japan

Bacterial strains were isolated from samples of Japanese rice vinegar (komesu) and unpolished rice vinegar (kurosu) fermented by the traditional static method. Fermentations have never been inoculated with a pure culture since they were started in 1907. A total of 178 isolates were divided into groups A and B on the basis of enterobacterial repetitive intergenic consensus-PCR and random amplified polymorphic DNA fingerprinting analyses. The 16S ribosomal DNA sequences of strains belonging to each group showed similarities of more than 99% with Acetobacter pasteurianus. Group A strains overwhelmingly dominated all stages of fermentation of both types of vinegar. Our results indicate that appropriate strains of acetic acid bacteria have spontaneously established almost pure cultures during nearly a century of komesu and kurosu fermentation.     

Production of citric acid with immobilized Aspergillus niger

Summary The spores of Aspergillus niger were entrapped in calcium-alginate beads and precultivated in growth media with various amounts of nitrogen. During the following citric acid production in shaking cultures an optimum of acid formation and yield was observed after the precultivation with 100–200 mg/l NH₄NO₃. The productivity of the immobilized Aspergillus was found to be 1.5 times higher than in the case of free pellets. The outgrowth of free mycelia into the medium could be provided by increasing the ratio particle-volume: medium volume, using a 1-l air-lift fermenter, by which means the productivity was increased twice as much as obtained in shaking culture.     

Characterization by plasmid profile analysis of acetic acid bacteria from wine, spirit and cider acetators for industrial vinegar production

The fermentation in industrial high acid submerged fermenters of wine, spirit or cider into vinegar was investigated by direct analysis of the plasmid patterns of the acetic acid bacteria involved. The strains were different in all samples. One strain from a red wine acetator was isolated by the double layer agar technique and proved to be the main fermenting organism. After inoculation of an acetator with this strain, the initiation of acidification was accelerated. Inoculation with spirit vinegar organisms led to immediate acidification. The spirit vinegar organisms were outnumbered after 12 d by fortuitously-occurring, highly adapted red wine vinegar bacteria. Over-oxidation was due to physiological regulation, a genetical change by plasmid loss or occurrence of new organisms was not observed.     

Submerged acetification of a vinegar base produced from waste pineapple juice

Submerged acetification experiments were performed to determine conditions necessary for the production of a satisfactory vinegar from waste pineapple juice using a pure culture inoculum. Vinegars containing up to 7%, by weight of acetic acid could be produced in less than 24 hr. with a conversion efficiency greater than 90% in both laboratory and pilot plant equipment. None of the bacterial nutrients added to the juice improved either the rate of production or the yield of acetic acid. Oxygen transfer during the process is discussed.