Methane fermentation of coastal mud sediment by a two-stage upflow anaerobic sludge blanket (UASB) reactor system

The removal of organic matter from a coastal mud sediment was carried out by a methane fermentation process under anaerobic conditions. In a batch acidogenic fermentation, the addition of vitamins containing thiamine, nicotinic acid and biotin dramatically enhanced acetate production from the mud sediment (200 g wet wt l(-1) artificial sea water), yielding 77 mM acetate after 6 days, which corresponded to 77% of the organic matter in the mud sediment, measured on the basis of chemical oxygen demand. Thereafter, the two-fold diluted, post-acidogenic fermentation liquor (PAF liquor) was continuously treated at 2.4x original dilution rate day(-1) for 30 days, using an upflow anaerobic sludge blanket methanogenic reactor containing the acclimated methanogenic sludge from the mud sediment. Acetate, 42 mM in the PAF liquor, was converted to methane at a maximum methane production rate of 96 mmol l(-1) day(-1); and 87.5% of the acetate and 88.7% of the total organic carbon in the PAF liquor were removed. Moreover, an efficient treatment of the mud sediment was carried out by a semi-continuous, two-stage reactor system, where the culture broth was circulated between acidogenic and methanogenic reactors. This two-stage reactor system gave a stable operation at 4-day intervals for one treatment period, yielding 112 mmol methane from the wet mud in the PAF liquor (278 g l(-1)).     

Enhanced ethyl caproate production of Chinese liquor yeast by overexpressing EHT1 with deleted FAA1

The fruity odor of Chinese liquor is largely derived from ester formation. Ethyl caproate, an ethyl ester eliciting apple-like flavor, is one of the most important esters in the strong aromatic Chinese liquor (or Luzhou-flavor liquor), which is the most popular and best-selling liquor in China. In the traditional fermentation process, ethyl caproate in strong aromatic liquor is mainly produced by aroma-producing yeast, bacteria, and mold with high esterification abilities in a mud pit at later fermentation stages at the expense of both fermentation time and grains rather than by the ethanol-fermenting yeast Saccharomyces cerevisiae. To increase the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae AY15) and shorten the fermentation period, we constructed a recombinant strain EY15 by overexpressing EHT1 (encoding ethanol hexanoyl transferase), in which FAA1 (encoding acyl-CoA synthetases) was deleted. In liquid fermentation of corn hydrolysate and solid fermentation of sorghum, ethyl caproate production by EY15 was remarkably increased to 2.23 and 2.83 mg/L, respectively, which were 2.97- and 2.80-fold higher than those of the parental strain AY15. Furthermore, an increase in ethyl octanoate (52 and 43 %) and ethyl decanoate (61 and 40 %) production was observed. The differences in fermentation performance between EY15 and AY15 were negligible. This study resulted in the creation of a promising recombinant yeast strain and introduced a method that can be used for the clean production of strong aromatic Chinese liquor by ester-producing S. cerevisiae without the need for a mud pit.     

Isolation of mesophilic solvent-producing clostridia from Colombian sources: physiological characterization, solvent production and polysaccharide hydrolysis

One hundred and seventy-eight new butanol-acetone producing bacteria related to saccharolytic clostridia were isolated from agricultural sources in Colombia and their fermentation potential was evaluated. Thirteen isolates produced more total solvents from glucose than Clostridium acetobutylicum ATCC 824. The isolates with the highest single solvent production were IBUN 125C and IBUN 18A with 0.46 mol butanol and 0.96 mol ethanol formed from 1 mol glucose, yielding 25. 2 and 29.1 g l(-1) total solvents, respectively, which is close to the maximum values described to date. Most of the new isolates produced exoenzymes for the hydrolysis of starch, carboxymethyl cellulose, xylan, polygalacturonic acid, inulin and chitosan. Together with the high efficiency of solvent production, these hydrolytic isolates may be useful for the direct fermentation of biomass. According to their physiological profile, the most solvent-productive isolates could be classified as strains of C. acetobutylicum, Clostridium beijerinckii, and Clostridium NCP262.     

Gas chromatography and gateway sensors for on-line state estimation of complex fermentations (butanol-acetone fermentation)

A fermentation system has been designed to demonstrate the use of gas chromatography (GC) for on-line monitoring of the butanol-acetone and other complex saccharolytic fermentations. Tangential flow ultrafiltration was used to sterilely and continuously obtain a cell-free filtrate from the fermentation broth for on-line GC analysis of butanol, butyrate, acetate, acetone, ethanol, and acetoin. The liquid injection system consists of a phosphoric acid contactor, a slider-type injection valve, and a heater to address the difficulties (ghosting) encountered in the analysis of carboxylic acids. The fermentation headspace gas was also analyzed by on-line GC for nitrogen and carbon dioxide, while hydrogen was measured by difference. Raw chromatographic data were analyzed by a chromatography data system. Both raw and processed data were transmitted to a VAX 11/750 computer for further processing (using the fermentation equation) and archiving. The fermentation equation, which has recently been derived and tested on completed fermentation data, was also found to be valid during transient fermentations and thus useful as a gateway sensor for calculating various fermentation parameters on-line. Such parameters include glucose concentration and gas composition, as well as a number of unobservable parameters (such as Y(ATP), excess ATP, and NAD reduced by FdH(2)), which characterize the state of the fermentation.     

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.     

L-天冬氨酸-β-脱羧酶发酵条件的响应面优化

利用Plackett-Burman设计和响应面分析方法对L-天冬氨酸-β-脱羧酶产生菌德阿昆哈假单孢菌XG-2-81的发酵条件进行优化.得出影响产L-天冬氨酸-β-脱羧酶的3个重要的因素为:玉米浆,多肽朊和装液量.通过对实验结果的方差分析,得到其最适条件分别为:玉米浆浓度0.64%,多肽朊浓度1.21%,装液量14.7...     

A media design program for lactic acid production coupled with extraction by electrodialysis

The aim of this study was to investigate industrial media for lactic acid fermentation to reduce the cost of nitrogen sources. Corn steep liquor (CSL) was successfully used at 5% (v/v) in batch fermentations. Use of soluble CSL improved the productivity approximately 20% with an advantage of clearer fermentation broth. Yeast extract (YE)-complemented CSL media further increased the productivity. It was found that 3.1 g L(-1) yeast extract and 5% CSL could be an effective substitute for 15 g L(-1) yeast extract in 10% glucose medium. Spent brewery yeast was also used as a sole nitrogen source equivalent to 5% CSL. Lactic acid was recovered by electrodialysis from the cell free broth. Depleted cell free broth supplemented with 5 g L(-1) of yeast extract performed reasonably in batch cultures. Reuse of the fermentation broth may reduce the cost of raw materials as well as minimize the fermentation wastes.     

纳豆激酶发酵液的脱色工艺研究

从11种待选脱色介质中筛选出330(OH)型树脂对纳豆激酶发酵液进行脱色研究.结果表明:(1)330(OH)型树脂对纳豆激酶发酵液中的色素为优吸型吸附,其动力学模型符合扩散方程,其动力学拟和方程为-ln(1-F)=0.0223t+0.0511,R2=0.9978,其中F=Qt/Qe,Qt为脱色时间为t时330(OH)型...     

Growth of Chaetomium cellulolyticum on Alkali-Pretreated Hardwood Sawdust Solids and Pretreatment Liquor

The treatment of a hardwood sawdust with 1% NaOH solution at 121°C dissolved 19.7% of the dry matter, mainly hemicellulose and lignin. Fermentation of the treated solids by Chaetomium cellulolyticum for 48 h gave a product containing 12.5% crude protein (total N × 6.25) on a dry weight basis. The in vitro rumen digestibility of the 48-h fermentation product was 30%, compared to 24% for the alkali-treated but unfermented sawdust. Growth was independent of sawdust particle size in the range 40 to 100 mesh. Fermentation of the pretreatment liquor gave a product containing up to 50% crude protein (dry weight basis) with an in vitro rumen digestibility of 65 to 76%. Approximately 6.7 g of crude protein was obtained from the treated solids and 2.2 g from the pretreatment liquor per 100 g of sawdust treated. The product from the pretreatment liquor fermentation has potential as a high-protein animal feed supplement but could not be produced economically without an outlet for the relatively indigestible product from the solids fermentation. Growth on the pretreatment liquor was strongly pH dependent; there was a considerable increase in the lag phase when the pH was lowered from 7.5 to 5.2. This effect appears to be due to an inhibitor whose toxicity is reduced at high pH.     

Fermentation to ethanol of pentose-containing spent sulphite liquor

Ethanolic fermentation of spent sulphite liquor with ordinary bakers' yeast is incomplete because this yeast cannot ferment the pentose sugars in the liquor. This results in poor alcohol yields, and a residual effluent problem By using the yeast Candida shehatae (R) for fermentation of the spent sulphite liquor from a large Canadian alcohol-producing sulphite pulp and paper mill, pentoses as well as hexoses were fermented nearly completely, alcohol yields were raised by 33%, and sugar removal increased by 46%. Inhibitors were removed prior to fermentation by steam stripping. Major benefits were obtained by careful recycling of this yeast, which was shown to be tolerant both of high sugar concentrations and high alcohol concentrations. When sugar concentrations over 250 g/L (glucose: xylose 70:30) were fermented, ethanol became an inhibitor when its concentration reached 90 g/L. However, when the ethanol was removed by low-temperature vacuum distillation, fermentation continued and resulted in a yield of 0.50 g ethanol/g sugar consumed. Further improvement was achieved by combining enzyme saccharification of sugar oligomers with fermentation. This yeast is able to ferment both hexoses and pentoses simultaneously, efficiently, and rapidly. Present indications are that it is well suited to industrial operations wherever hexoses and pentoses are both to be fermented to ethanol, for example, in wood hydrolysates.     

响应面法优化赖氨酸发酵培养基

运用Ｂｏｘ－Ｂｅｈｋｅｎ设计的响应面试验（Ｒｅｓｐｏｎｓｅ－ｓｕｒｆａｃｅ ｅｘｐｅｒｉｍｅｎｔ）对黄色短杆菌（Ｂｒｅｕｉｂａｃｔｅｒｉｕｍ ｆｌａｕｕｍ）ＦＢ４２进行赖氨酸发酵的培养基进行了优化。响应面法对７２小时分批发酵结果的优化分析表明：硫酸铵、豆饼水解液及玉米浆三种因素对赖氨醚发酵无明显的交互作用,在硫酸铵、豆饼水解液和玉米浆的浓度分别为５％、２％和３％时,发酵液中的产酸达到５４．０６ｇ／     

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%。     

芝麻香型白酒发酵过程中乳酸菌多样性及其演替规律

【背景】乳酸菌是白酒发酵过程中一类非常重要的微生物,其种类及动态变化对于白酒品质具有重要影响。然而,目前对于芝麻香型白酒发酵过程中乳酸菌群落结构及其演替规律的认识并不全面。【目的】揭示芝麻香型白酒发酵过程中乳酸菌的多样性及菌群的演替规律,为更好地探索白酒酿造机理和控制白酒品质提供生物学依据。【方法】利用高通量测序技术对芝麻香型白酒发酵过程中乳酸菌菌群演替进行跟踪分析,同时采用实时荧光定量PCR对发酵过程中乳酸菌的生物量进行定量分析。【结果】高通量测序结果显示,芝麻香型白酒发酵过程涉及5个属的乳酸菌:魏斯氏菌属(Weissella)、片球菌属(Pediococcus)、乳杆菌属(Lactobacillus)、明串珠菌属(Leuconostoc)和乳球菌属(Lactococcus),共计43种乳酸菌。其中,在发酵过程中平均相对丰度大于0.5%的乳酸菌有10种,分别是类肠膜魏斯氏菌(Weissella paramesenteroides)、食窦魏斯氏菌(Weissella cibaria)、融合魏斯氏菌(Weissella confusa)、戊糖片球菌(Pediococcus pentosaceus)、假肠膜明串珠菌(Leuconostoc pseudomesenteroides)、发酵乳杆菌(Lactobacillus fermentum)、植物乳杆菌(Lactobacillus plantarum)、副干酪乳杆菌(Lactobacillus paracasei)、耐酸乳杆菌(Lactobacillus acetotolerans)和Lactobacillus sp.。在堆积发酵过程中,Weissella属占细菌总量的50%以上,其次是Pediococcus属和Lactobacillus属,而Leuconostoc属和Lactococcus属相对较少。在窖池发酵过程中Lactobacillus属的乳酸菌逐渐成为优势细菌,尤其是Lactobacillus sp.在窖池发酵中后期相对丰度达到80%以上。实时荧光定量PCR结果显示,在堆积发酵和窖池发酵前期乳酸菌总量变化不大;从窖池发酵5 d开始,乳酸菌总量迅速上升,30 d时达到最大值。【结论】对白酒发酵过程中乳酸菌种类及动态变化的研究有助于探究白酒酿造过程中乳酸菌功能,进而解析白酒酿造机理,最终达到控制白酒品质的目的。     

L-lactic acid production by Lactobacillus casei fermentation with corn steep liquor-supplemented acid-hydrolysate of soybean meal

Batch and fed-batch fermentation studies were performed to evaluate the potential of corn steep liquor (CSL)-supplemented acid-hydrolysate of soybean meal (AHSM) as an alternative to yeast extract (YE) for the production of L-lactic acid by Lactobacillus casei LA-04-1. The CSL-supplemented AHSM gave an outstanding result in supporting L-lactic acid production from glucose. In the exponential fed-batch fermentation, the concentration, yield and productivity of L-lactic acid were 162.5 g/L, 89.7% and 1.69 g/L per h, respectively, which were lower than those with 20 g/L YE (180 g/L, 90.3%, 2.14 g/L per h) after 96 h of fermentation. However, the raw material cost of the nitrogen resource was estimated as only 25% of that using the YE.     

一株芽孢杆菌在维生素C二步发酵中对小菌的促进作用

从土壤中分离到1株能更好促使小菌生长和产酸的芽孢杆菌B601,作为伴生菌与巨大芽孢杆菌相比,在生长过程中,发酵液中B601活菌数小于巨大芽孢杆菌,而其芽孢数则多于巨大芽孢杆菌。对B601组成菌系的发酵条件进行优化,得到如下结果：100g／L L-山梨糖、6g／L尿素、10g/L玉米浆、培养温度30℃和发酵周期44h。与巨大芽孢杆菌组成菌系相比其底物,L-山梨糖质量浓度提高了25％,尿素下降了50％．玉米浆质量浓度下降了33％,温度提高了2℃,发酵周期缩短了4h。结果表明：B601作为伴生菌,与巨大芽孢杆菌相比,该菌株明显提高了发酵效率。     

中国酱香型白酒酿造酿酒酵母的独特生理代谢特征

【目的】酿酒酵母是白酒发酵的主要微生物,对白酒的产量及质量都有重要影响。而酱香酒酿造环境具有高温、酸性、高乙醇等胁迫因素,研究其中酿酒酵母的生理代谢特征并有目的地应用于白酒实际生产中的意义。【方法】从酱香酒酿造环境中筛选一株性能优良的酿酒酵母菌株,比较其与酿酒酵母模式菌株S288c和商业酵母的生理代谢特征。【结果】从酱香型酒醅中筛选得到性能优良的Saccharomyces cerevisiae MT1,该菌株可耐受高温42 °C,高浓度乙醇(16%,体积比),低pH (2.0),其最大比生长速率和最大比产乙醇速率分别达到了S288c的125%和114%,其乙醇转化率也要高于其他菌株。一些挥发性物质只有在MT1的发酵液中可检测到,包括苯并噻唑、2,3-二氢苯并呋喃、4-乙烯基愈创木酚以及丁羟甲苯等,MT1的苯乙醇、法呢醇、橙花叔醇、乙偶姻和大马酮量也要高于S288c。另外,MT1可以利用多种碳源发酵产乙醇,如半乳糖、麦芽糖、蜜二糖、松二糖、海藻糖和棉子糖等。【结论】来源于酱香酒酿造的S. cerevisiae MT1具有高耐受力、高效的发酵性能及更广泛的碳源利用图谱,并能生成多种挥发性物质。     

ILV2基因缺失对啤酒酵母生长与双乙酰代谢的影响

【目的】旨在应用分子生物学方法降低啤酒发酵液中双乙酰含量,改善啤酒感官质量。【方法】以酿酒酵母S2(Saccharomyces cerevisiae)为出发菌株,通过同源重组敲除四倍体啤酒酵母α-乙酰乳酸合成酶部分基因(ILV2),构建缺失一个和两个ILV2等位基因的突变株QI2-1和QI2-2,并进行啤酒发酵实验。【结果】ILV2基因的缺失,会导致菌株初始生长速率的降低。其中QI2-2较为明显,12 h时,突变株与出发菌株的生长速率达到一致。啤酒发酵结果表明,与出发菌株相比,突变株QI2-1双乙酰峰值与双乙酰最终含量分别降低17.50%和17.83%,而QI2-2分别降低51.67%和45.65%。其他啤酒指标如酒精度、发酵度、残糖和风味物质等略有变化,但都在优质啤酒指标范围内,符合啤酒发酵的质量要求。【结论】通过同源重组敲除部分ILV2基因和选育低产双乙酰菌株是降低啤酒双乙酰含量、提高啤酒质量的有效方法,具有一定的实际应用价值。     

响应面分析法优化黑根霉胞外多糖发酵工艺

实验以商品化的马铃薯葡萄糖液体培养基为基础培养基,以胞外粗多糖产量为考察指标,运用响应面分析法考察玉米浆浓度、KH₂PO₄浓度和发酵时间3个因素对胞外多糖发酵产量的影响,以获得黑根霉胞外多糖发酵最优工艺,建立高产、稳定、可控的胞外多糖发酵生产工艺技术方案。经响应面分析,各因素按照对响应值的影响顺序为：玉米浆浓度>发酵时间> KH₂PO₄浓度,且玉米浆浓度、发酵时间对胞外多糖产量的影响极显著,KH₂PO₄浓度对胞外多糖产量的影响不显著。胞外多糖发酵最优工艺为：玉米浆3.2mg/mL、KH₂PO₄ 1.5mg/mL和发酵时间132h,在此条件下胞外多糖的最大预测产量为0.824mg/mL。实验重复性好,是一个高产、稳定、可控的胞外多糖发酵生产工艺技术方案,可以指导黑根霉胞外多糖发酵。     

利用肌苷发酵废液生产单细胞蛋白的研究

我国在利用微生物降解酒精废液、造纸厂废液和味精厂废液等方面已有许多报道,但是,利用肌苷发酵废液作为底物进行单细胞生产还未见报道。同时,提取肌苷后的发酵废液,其营养成份仍很丰富,其中,还原糖含量约为2％,总氮含量为300mg/L～400mg/L,以及一些无机盐。COD值高达186276mg/L。目前,一般肌苷生产厂都将废液排放掉,这不仅严重地污染了环境,而且造成很大的浪费。有鉴于此,本课题进行了菌种的筛选和驯化以及工艺方面的试验。     

清香型白酒发酵过程中酵母群落结构及其与尿素代谢的关系

【目的】探索清香型白酒发酵过程中酵母群落结构及演变,分析潜在的关键尿素代谢酵母及其环境调控因素,为降低发酵过程中氨基甲酸乙酯的含量提供理论依据。【方法】通过相关性分析明确发酵过程中氨基甲酸乙酯主要前体物质及其代谢微生物类群,利用高通量测序技术解析酵母群落结构组成,并结合偏最小二乘回归分析寻找潜在的关键尿素代谢酵母。采用冗余分析评价发酵过程中环境因素对酵母群落结构的影响。【结果】尿素是清香型白酒发酵过程中氨基甲酸乙酯的主要前体物质,酵母是尿素代谢的主要微生物。高通量测序结果显示,在97%的相似度下进行操作分类单元聚类后,共鉴定出22个酵母种。其中,嗜高压有孢汉生酵母(Hanseniaspora osmophila)、发酵毕赤酵母(Pichia fermentans)和酿酒酵母(Saccharomyces cerevisiae)与尿素合成存在正相关性,库德毕赤酵母(Pichia kudriavzevii)与尿素降解存在正相关性。酒醅含水量、p H、乙醇和精氨酸是影响发酵过程中酵母群落演替的重要环境因素。【结论】环境因素影响潜在的关键尿素代谢酵母,为降低发酵过程中尿素与氨基甲酸乙酯含量提供理论依据。