洋河浓香型白酒发酵过程酒醅微生物群落结构解析及其与有机酸合成的相关性

【目的】解析江苏洋河酒厂浓香型白酒窖内发酵过程酒醅微生物群落结构,建立酒醅微生物与主要有机酸合成的关联性。【方法】通过宏基因组测序获得白酒发酵过程中微生物群落结构变化规律,利用主成分分析和偏最小二乘回归分析寻找酒醅中影响主要有机酸合成的关键微生物。【结果】根据微生物组成结构变化和有机酸合成变化规律,可将白酒窖内发酵分为两个时期(0–14 d和15–60 d)。其中窖内发酵0–15 d与主要有机酸合成相关的微生物数量显著高于15–60 d的。窖内发酵过程与主要有机酸合成相关的微生物包括7个菌属,分别为乳杆菌属(Lactobacillus)、葡萄球菌属(Staphylococcus)、酵母属(Saccharomyces)、Naumovozyma、伊萨酵母属(Issatchenkia)、嗜冷芽孢杆菌属(Psychrobacillus)和根霉属(Rhizopus)。【结论】本研究识别了白酒窖内发酵过程中与主要有机酸合成相关的核心和关键微生物,可为阐明白酒窖内发酵产酸机理和保障白酒品质的稳定性奠定研究基础和理论依据。     

Fed-batch fermentation with and without on-line extraction for propionic and acetic acid production byPropionibacterium acidipropionici

Fed-batch propionic and acetic acid fermentations were performed in semi-defined laboratory medium and in corn steep liquor withPropionibacterium acidipropionici strain P9. On average, over four experiments, 34.5 g/l propionic acid and 12.8 g/l acetic acid were obtained in about 146 h in laboratory medium with 79 g/l glucose added over five feeding periods. The highest concentration of propionic acid, 45 g/l, was obtained when the glucose concentration was not allowed to drop to zero. In corn steep liquor 35 g/l propionic acid and 11 g/l acetic acid were produced in 108 h from 59.4 g/l total lactic acid provided as seven feedings of corn steep liquor. Extractive fed-batch fermentations were conducted in semi-defined medium using either flat-sheet-supported liquid membranes or hollow-fiber membrane extraction to remove organic acids from the culture medium. As operated during the course of the fermentation, these systems extracted 25% and 22% of the acetic acid and 36.5% and 44.5% of the propionic acid, respectively, produced in the fermentation. Total amounts of acids produced were about the same as in comparable nonextractive fermentations: 30–37 g/l propionic acid and 13 g/l acetic acid were produced in 150 h. Limitations on acid production can be attributed to limited substrate feed, not to failure of the extraction system.Journal paper J-16303 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 3122.     

Fermentation of kraft black liquor for the production of citric acid by Candida tropicalis

Summary The production of citric acid by batch fermentation with the yeast strain Candida tropicalis ATCC 20240 was chosen as a potential process for the valorization of kraft black liquor. The effect of nitrogen concentration was studied and direct bioconversion of acetate to citrate was achieved when no nitrogen was supplemented to the medium. The use of kraft black liquor's acetate as a potential substrate for citric acid production was investigated. The acid precipitated liquor was highly inhibitory when its concentration was above 25% of the fermentation broth content. The yields of citric acid at low concentrations of kraft black liquor (5% and 15%) were the same as those recorded in synthetic acetate medium. Other organic acids present in the liquor may affect the yields and rates of citric acid production over acetate. Substrate uptake rates and product formation rates were lower, however, in comparison to synthetic media. The utilization of immobilized biomass improved the process parameters on kraft black liquor and enhanced the fermentation capabilities.     

Comparison of corn steep liquor with other nutrients in the fermentation of D-Xylose by Pichia stipitis CBS 6054

Summary Pichia stipitis CBS 6054 ferments D-Xylose to ethanol in a medium containing corn steep liquor as the only source of nitrogen, amino acids, vitamins and other nutrients. The ethanol yield and fermentation rate compare favorably to those obtained with media containing more expensive sources of nitrogen, vitamins and amino acids. Corn steep liquor is a good source of nutrients that can support growth and fermentation activity of this xylose fermenting yeast.     

两步发酵过程中有机酸对产1，3-丙二醇的影响

考察了基因工程菌发酵生产1.3 丙二醇过程中,有机酸对发酵过程的影响,并选用了不同的离子交换树脂对甘油发酵液进行处理。发现有机酸、特别是乳酸对1.3丙二醇生产的抑制作用最明显。在使用离子交换树脂处理有机酸的过程中,确定了使用005号离子交换树脂处理效果最好,005号离子交换树脂可除去大部分的有机酸,处理后的发酵液发酵产1.3丙二醇产量比未处理的发酵液产量提高166%,转化率提高34%。     

Production of a herbicide, 5-aminolevulinic acid,by Rhodobacter sphaeroides using the effluent of swine waste from an anaerobic digestor

Summary For the production of a herbicide, 5-amino-levulinic acid (ALA), from anaerobic digestion liquor, the utilization of the photosynthetic bacterium, Rhodobacter sphaeroides was examined. This bacterium could produce ALA extracelularly from this liquor with the addition of levulinic acid (LA), an inhibitor of ALA dehydratase (ALAD), and glycine, a precursor of ALA biosynthesis in the Shemin pathway. Succinate (another precursor) addition was unnecessary for ALA production. When repeated additions of LA were made together with glycine ALA production was significantly enhanced. However, above three additions of LA, ALA production was not further enhanced. The maximum value of ALA production attained was 4.2 mM (0.63 g/ 1), which was over double that of other ALA producers such as Chlorella vulgaris. Propionic acid was predominantly utilized compared with other lower fatty acids, suggesting that this might be converted to ALA via succinyl-coenzyme A (CoA) in the methylmalonyl-CoA pathway.Offprint requests to: Y. Nishizawa     

Mixed-solid substrate fermentation. A novel process for enhanced lipase production by Candida rugosa

Candida rugosa was cultivated in a mixed-solid substrate containing coconut oil cake (COC) and fine and coarse wheat bran (1:1:1) with an initial water activity (a_w) of 0.92. The substrate was modified by adding a mineral solution (5%), corn steep liquor (6%), maltose (2%), peptone (3%), olive oil (10%), gum arabic (0.4%), different fatty acids (0.3%) and Tweens (0.5%). Fermentation in a column fermenter significantly improved the lipase yield to 118.2 Units per gram of dry fermented substrate [U/gds] at 72 h. This result was obtained 24 hours earlier than in our former studies (87.76 U/gds at 96 h) in COC, and the yield showed a 38% increase. Growth was measured indirectly by determining the glucosamine content in the cell wall of the yeast contained in the fermented matter, after its hydrolysis.     

Leaching of iron from kaolins by a spent fermentation liquor: Influence of temperature,pH, agitation and citric acid concentration

Summary The efficiency of a biological bleaching process on a highly contaminated iron oxides kaolin was analyzed. The bio-bleaching method consists of two steps: first, the fermentation of a chemically-defined medium byAspergillus niger leads to a spent liquor which, in a second step, is employed as leaching agent for kaolin. In the leaching process 43% of iron oxides was removed when the kaolin was treated at 60°C for 5 h and the whiteness index was increased to 67%. Extracellular enzymes were not responsible for leaching, which is basically due to organic acids produced during the fermentation.     

Critical factors in chitin production by fermentation of shrimp biowaste

Factors affecting Lactobacillus fermentation of shrimp waste for chitin and protein liquor production were determined. The objective of the fermentation is medium conditioning by Lactobacillus through production of proteases and lowering of the pH. The efficiency was tested by conducting fermentation of biowaste in 1-l beakers with or without pH adjustment using different acids. Addition of 5% glucose to the biowaste supported the growth of lactic acid bacteria and led to better fermentation. Among four acids tested to control pH at the start and during fermentation, acetic acid and citric acid proved to be the most effective. In biowaste fermented with 6.7% L. plantarum inoculum, 5% glucose, and pH 6.0 adjusted with acetic acid, 75% deproteination and 86% demineralization was achieved. Replacement of acetic acid by citric acid gave 88% deproteination and 90% demineralization. The fermentation carried out in the presence of acetic acid resulted in a protein fraction that smelled good and a clean chitin fraction. Received: 4 April 2000 / Received revision: 9 June 2000 / Accepted: 9 June 2000     

Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidipropionici

Cells of the propionate-tolerant strain Propionibacterium acidipropionici P200910, immobilized in calcium alginate beads, were tested for propionic and acetic acid production both in a semidefined laboratory medium and in corn steep liquor in batch, fed-batch, and continuous fermentation. Cell density was about 9.8 × 10⁹ cells/g (wet weight) of beads, and beads were added to the medium at 0.1 g (wet weight) beads/ml. Beads could be reused for several consecutive batch fermentations; propionic acid production in the tenth cycle was about 50%–70% of that in the first cycle. In batch culture complete substrate consumption (glucose in semidefined medium, lactate in corn steep liquor) and maximum acid production were seen within 36 h, and acid yields from the substrate were higher than in free-cell fermentations. Fed-batch fermentations were incubated up to 250 h. Maximum propionic acid concentrations obtained were 45.6 g/l in corn steep liquor and 57 g/l in semidefined medium; this is the highest concentration achieved to date in our laboratory. Maximum acetic acid concentrations were 17 g/l and 12 g/l, respectively. In continuous fermentation of semidefined medium, dilution rates up to 0.31 h^–1 could be used, which gave higher volumetric productivities (0.96 g l^–1 h^–1 for propionic acid and 0.26 g l^–1 h^–1 for acetic acid) than we have obtained with free cells. Corn steep liquor shows promise as an inexpensive medium for production of both acids by immobilized cells of propionibacteria.Journal paper no. J- 15614 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project no. 3122     

Fed-batch and continuous fermentation ofSelenomonas ruminantium for natural propionic,acetic and succinic acids

An anaerobic fermentation process was developed for production of natural propionic, acetic and succinic acids froml-lactic acid usingSelenomonas ruminantium. Thel-lactic acid was quickly converted to a racemic mixture and there was no enantiomeric preference for further metabolism. The lactic acid was metabolized to propionic, acetic and succinic acids typically in a molar ratio of about 531. However, the ratio of propionate: succinate started high (as much as 221), before declining to as low as 51 after the first 48 h. Nutrients in corn steep liquor and yeast extract were necessary for optimal production of propionic acid. The corn steep liquor and yeast extract were heat stable at neutral pH, but some nutritional qualities were lost when heated at pH 2.4. In fed-batch fermentation on lactic acid 2.0% propionic acid was produced in 48 h and 2.3% in 68 h. A continuous culture operated at a dilution rate of 0.055 h^–1 and a lactic acid feed concentration of 30 gL^–1 had a propionic acid productivity of 0.59 gL^–1h^–1. The steady state results were: lactic acid 0.6%, propionic acid 1.1%, acetic acid 0.50%, and succinic acid 0.33%.     

Bacterial decolorization of black liquor in axenic and mixed condition and characterization of metabolites

The pulping byproducts (black liquor) cause serious environmental problem due to its high pollution load. In order to search the degradability of black liquor, the potential bacterial strains Citrobacter freundii (FJ581026) and Citrobacter sp. (FJ581023) were applied in axenic and mixed condition. Results revealed that the mixed bacterial culture are more effective than axenic condition and can reduce 82% COD, 79% AOX, 79% color and 60% lignin after 144 h of incubation period. Additionally, the optimum activity of lignin degrading enzyme was noted at 96 h and characterized as manganese peroxidase (MnP) by SDS–PAGE analysis. Further, the HPLC analysis of control and bacterial degraded sample has shown the reduction as well as shifting of peaks compared to control indicating the degradation as well as transformation of compounds of black liquor. The comparative GC–MS analysis of control and degraded black liquor revealed that along with lignin fragment some chlorophenolic compounds 2,4,6-trichlorophenol, 2,3,4,5-tetrachlorophenol and pentachlorophenol were detected in black liquor degraded by axenic culture whereas these chlorophenolic compounds were completely absent in black liquor degraded by mixed bacterial culture. These chlorophenol inhibit the oxidative degradation which seems a major reason behind the low degradability of axenic degradation compared to mixed culture. The innovation of this aerobic treatment of alkaline black liquor opens additional possibilities for the better treatment of black liquor along with its metabolic product.     

Growth ofAureobasidium pullulans on waste water hemicelluloses

Strain Aureobasidium pullulans capable of utilizing hemicelluloses and xylan was cultivated on processed waste dialysis liquor from the production of viscose fibres, containing about 1.5% hemocelluloses. Basic conditions of biomass production were tested on a laboratory scale. The dialysis waste liquor adjusted with mineral acids to pH 4--5 and supplemented with 0.05% yeast autolyzate and 0.2% ammonium sulphate affords protein yields of about 0.8 g/l, corresponding to 4.0--4.5 g dry biomass. Biomass is isolated together with residual water-insoluble hemicelluloses which are not utilized by the microorganism. The total utilization of hemicelluloses attains about 70%.     

Gel Filtration of Lignosulphonic Acids and Peptide-Precipitating Abilities of the Separated Fractions

Lignosulphonic acids in dialysed sulphite spent liquor and purified lignosulphonic acids were subjected to gel chromatography on Sephadex G-75, G-100 and G-200 and the fractions tested for peptide-precipitating ability. About 56 % of the total lignosulphonic acids in the dialysed sulphite spent liquor had estimated molecular weights above 90000 and about 72 % above 44000. About 94 % of the purified lignosulphonic acids had molecular weights above 90000 and the remaining 6 % had above 36000. The major peptide-precipitating activity of the lignosulphonic acids was due to fractions with molecular weights in excess of 90000. The percentage of peptides in the peptide-lignosulphonic acid precipitates was found to be 80–90. The molecular weights of the peptides used were found to have an upper limit of about 20000. The lower limit for molecular weights of lignosulphonic acid-precipitating peptides is estimated to be below 6000.     

Solubilization of cobalt from ocean nodules at neutral pH—a novel bioprocess

A marine organism (Bacillus M1) isolated from Indian Ocean manganese nodules was characterized. The organism grew well in artificial seawater medium, at near neutral pH, 30°C and 0.25 M NaCl, and showed MnO₂-reducing activity. Growing cultures of Bacillus M1 as well as cell-free spent liquor from fully-grown cultures were employed to extract metals from the nodules. The spent liquor of cultures of the organism could dissolve around 45% cobalt (Co) at a pH of 8.2 in 2 h. Co recovery by this treatment was comparable to that in acidic leaching with 2.5 M hydrochloric acid solutions, and was independent of pulp density (w/v ratio). The amount of Co dissolved was beyond the thermodynamic solubility limit in aqueous solution at a pH of 8.2. It is inferred that the metabolites present in the spent liquor played a pivotal role in complexing the Fe (III) phase, solubilizing Co in the process. Partial characterization of spent liquor by spot tests, UV visible spectroscopy and FTIR spectroscopy, showed the presence of siderophore-like phenolic compound(s) with an attached carboxyl group that might form soluble organic complexes with Fe (III).     

Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21

Culture conditions of Schizochytrium limacinum SR21 for the purpose of microbial docosahexaenoic acid (DHA) production were investigated. The strain SR21 showed a wide tolerance to salinity; that is, the optimum salinity was between 50% and 200% that of sea water. Monosaccharides (glucose and fructose) and glycerol supported good cell growth and DHA yield. Di- and polysaccharides, oleic acid, and linseed oil gave low DHA yields. A high content of DHA (more than 30% of total fatty acids) was obtained from culture on glucose, fructose, and glycerol, and also the strain had simple polyunsaturated fatty acid profiles. The major polyunsaturated fatty acids other than DHA were n-6 docosapentaenoic acid only, and the contents of icosapentaenoic acid and arachidonic acid were less than 1%. Using corn steep liquor as a nitrogen source, a high total fatty acid content was obtained. The total fatty acid content in the dry cell weight increased as the concentration of the nitrogen source decreased, reached more than 50%. An increase in carbon source concentration led to a high DHA yield. A maximum DHA yield of more than 4 g/l was obtained in both glucose and glycerol media at 9% and 12% respectively. S. limacinum SR21 was thought to be a promising resource for microbial DHA production yielding a good level of productivity as well as a simple polyunsaturated fatty acid profile. Received: 26 June 1997 / Received revision: 29 August 1997  / Accepted: 19 September 1997     

Design of an efficient fermentation process for the production of Metarhizium acridum blastospores

Using a sequential approach, we described efficient blastospore production in a stirred tank bioreactor (3?L capacity). We used the response surface methodology to optimise the media ingredients and fermentation parameters to obtain the maximum production of blastospores by a locally collected isolate of Metarhizium acridum (Ascomycota: Hypocreales). The results showed that a liquid culture medium supplemented with monopotassium phosphate (15.17?g/L), corn steep liquor (69.25?g/L), and casamino acids (80.68?g/L) in a stirred tank bioreactor under operating conditions constant at 635?rpm, a temperature of 26°C, and pH 3.3 produced 1.25?×?10⁸?blastospores (bls)/ml, with 93% viability after 120?h of fermentation. This bioreactor yield compares favourably with the yields obtained by shake flask production and confirms the suitability of the media and production parameters for the potential scale-up fermentation production of M. acridum.     

Utilization of volatile fatty acids from the anaerobic digestion liquor of sewage sludge for 5-aminolevulinic acid production by photosynthetic bacteria

Using volatile fatty acids (VFA) from the anaerobic digestion liquor of sewage sludge, up to 9.2 mm 5-aminolevulinic acid (ALA) could be produced by Rhodobacter sphaeroides under anaerobic-light (5 kLux) conditions with repeated addition of levulinic acid (LA) and glycine and using a large inoculum (approx. 2 g/l of cells, initially from glutamate/malate medium). As the VFA medium also contained organic nitrogen sources such as glutamic acid, the cells were later grown up to about 2 g/l in the VFA medium instead of the glutamate/malate medium. ALA production was then again promoted by adding LA and glycine. Using this improved method, up to 9.3 mm ALA was produced by feeding propionate and acetate together with LA and glycine, indicating that VFA medium formed from sewage sludge could be useful for ALA production.     

Production of D-Alanine by Corynebacterium fascians

A strain identified as Corynebacterium fascians was found to accumulate extracellular D-alanine from glycerol. Cultural conditions for the accumulation of D-alanine were investigated and, as a result, a yield of 7 g of D-alanine per liter was obtained after a 96-h incubation in a medium containing 5% glycerol, 4% (NH(4))(2)HPO(4), and 0.3% corn steep liquor. Optical purity of D-alanine was dependent upon the concentration of corn steep liquor. At the optimal condition, almost optically pure D-alanine was formed and readily isolated (5 g/liter) from the fermentation broth. The product was not contaminated with any detectable amount of other amino acids, except for glycine which was present at a concentration of less than 1 percent.     

Pretreatment Of Hardwood (Eucalyptus Regnans) Sawdust By Autohydrolysis Explosion And Its Saccharification By Trichodermal Cellulases

Autohydrolysis explosion pretreatment of hardwood (Eucalyptus regnans) sawdust at 200°C and 6.9 MPa gas pressure (steam + nitrogen) for 5 min solubilized 85% of the total hemicellulose components and produced a pulp that was highly accessible to attack by cellulases from Trichoderma reesei C-30 and by a commercial preparation, Meicelase. The autohydrolysis liquor, representing 15% of the original weight of the sawdust on a solids basis, consisted mainly of xylose, xylose oligomers and minor amounts of galactose, mannose, arabinose, glucose and uronic acids. Enzymic hydrolysis of pretreated E. regnans pulps using Trichodermal cellulases resulted in saccharification yields of <50% within 24 h from 10% (w/v) substrate slurries and 20 cellulase (FPU) units per g of pretreated pulp. The cellulose-to-glucose conversions were lower and this was attributable to the production of a compound(s) during enzymic hydrolysis that was inhibitory to the β-glucosidase component, but not the cellulases, in the Trichodermal cellulase preparations. Enzymic digests supplemented with Novozym 188 β-glucosidase showed >70% cellulose-to-glucose conversion within 24 h under similar conditions of hydrolysis. The inhibitor compound was not inhibitory to the Novozym 188 β-glucosidases. Alkali-extracted autohydrolysis-exploded pulps were less susceptible to hydrolysis than unextracted pulps. Factors that influenced the extent of cellulose conversion into glucose such as enzyme-substrate and cellulase-to-β-glucosidase ratios are also discussed.