固定化细胞利用离子交换树脂萃取发酵乳酸的研究

本文提出了利用海藻酸钙凝胶包埋固定化乳酸菌生产乳酸,用离子交换树脂从发酵液中分离出乳酸的新方法。该法成功地消除了产物乳酸对乳酸菌生长和产物乳酸形成的抑制作用,使发酵时间由１２０小时缩短到９６小时,乳酸的体积生产率由０．３２８ｇ／Ｌ·ｈ提高到０．４３２ｇ／Ｌ·ｈ。     

乳杆菌对致病性大肠杆菌K88和O138体外存活抑制的动力学研究

研究了一株分离自断奶仔猪小肠黏膜的肠乳杆菌L1（Lactobacillus intestinalis）体外发酵特性,及其代谢产物对病原性大肠杆菌Escherichia coli K88和O138存活的影响。体外发酵结果表明：发酵12h后,L,菌液pH值迅速降至3．90,并产生大量乳酸,为104．08mmol／L。L1菌株代谢产物对K88和O138体外生长抑制的动力学研究表明：L1菌株代谢产物对K88和O138存活具有很强的抑制作用;L1菌株发酵液与含相同浓度乳酸的自制培养液比较结果表明：乳酸在L1菌株代谢物对K88和O138存活抑制中发挥了主要作用：K88和O138对pH4．5的MRS培养液具有一定的耐受能力。     

1,3-丙二醇发酵过程中乳酸抑制的研究

目的:研究乳酸对克雷伯氏肺炎杆菌(Klebsiella pneumonia)产1,3-丙二醇的影响。方法:通过在摇瓶和反应器水平下分析不同菌株(包含无乳酸、2,3-丁二醇产生的基因敲除菌)的乳酸代谢特性。结果:前期添加6 g/L的乳酸使1,3-丙二醇的产量降低了19%,而发酵10h后添加乳酸几乎不表现出抑制作用。通过对乳酸敲除菌株的代谢分析发现,发酵后期能够消耗培养基中的乳酸,这在一定程度上也反映了菌体发酵后期对乳酸的耐受性。结论:乳酸的抑制作用主要发生在1,3-丙二醇发酵的前期。解除了一株无副产物2,3-丁二醇生产株前期乳酸的过早积累后,1,3-丙二醇的的产量提高了56%。     

木质纤维素酸解副产物对D-乳酸生产菌Sporolactobacillus sp.Y2-8生长及发酵的影响

选择乙酸根、糠醛、5-羟甲基糠醛、苯酚、香草酸和丁香醛等6种典型木质纤维素酸解副产物,考察它们对D-乳酸生产菌Sporolactobacillus sp.Y2-8生长及发酵的影响。实验结果表明:酚类物质抑制作用最强烈,0.25 g/L丁香醛已经完全抑制了菌体的生长和D-乳酸的发酵;苯酚和香草酸在低浓度(≤1.0 g/L)时抑制作用较小,但质量浓度达到3 g/L时对D-乳酸产量的抑制率分别为99%和70%;3 g/L糠醛和5-羟甲基糠醛对产物的抑制率分别为60%与20%,抑制作用小于酚类;乙酸根的影响最小,10 g/L的乙酸钠对菌体的生长和发酵几乎无抑制作用;当抑制物混合时,存在着相互促进作用,抑制作用更强烈。     

乳杆菌对致病性大肠杆菌K88和O138体外存活抑制的动力学研究

研究了一株分离自断奶仔猪小肠黏膜的肠乳杆菌L1(Lactobacillus intestinalis)体外发酵特性,及其代谢产物对病原性大肠杆菌Escherichia coli K88和O138存活的影响.体外发酵结果表明:发酵12 h后,L1菌液pH值迅速降至3.90,并产生大量乳酸,为104.08 mmol/L.L1菌株代谢产物对K88和O138体外生长抑制的动力学研究表明:L1菌株代谢产物对K88和O138存活具有很强的抑制作用;L1菌株发酵液与含相同浓度乳酸的自制培养液比较结果表明:乳酸在L1菌株代谢物对K88和O138存活抑制中发挥了主要作用;K88和O138对pH 4.5的MRS培养液具有一定的耐受能力.     

有机酸胁迫下厌氧污泥产氢效果

乙酸、丙酸、乳酸及丁酸是厌氧发酵产氢过程中4种主要的液相末端发酵产物,其积累对产氢过程有一定的抑制作用。实验利用多种有机酸胁迫提高污泥的酸耐受能力,并以污泥中脱氢酶活性为生化指标,对不同浓度酸胁迫下厌氧污泥活性变化进行了研究。通过对胁迫后污泥产氢量及末端产物进行对比。结果表明,酸胁迫后污泥产氢量有一定增加,其中乙酸和丁酸胁迫效果最好,较对照组提高了近一倍;末端产物分析研究表明,不同的有机酸胁迫后,其产量在发酵过程中都有一定的增加,而乙酸含量在酸胁迫后都有不同程度的提高。     

清香型白酒中乳酸菌和酵母菌的相互作用

【目的】探究清香型白酒中不同乳酸菌和酵母菌的相互作用,了解不同菌株的发酵性能,为更深入地认识白酒发酵机理、实现发酵过程优化提供理论基础。【方法】利用程序控温和固态发酵模拟清香型白酒酿造环境,测定纯培养和共培养中菌株的理化指标、活菌数以及主要代谢产物的变化。【结果】Saccharomyces cerevisiae YJ1糖消耗快产乙醇和酯类物质多,Lactobacillus plantarum JMRS4糖消耗快产酸较多。共培养中乳酸菌对Saccharomyces cerevisiae YJ1的生长和产乙醇抑制较大,对Candida aaseri MJ7产乙醇几乎无影响。乳酸菌对Pichia kudriavzevii MJ14的生物量和乙醇代谢抑制作用较小,还对其产己酸乙酯、乙酸乙酯和异戊醇等代谢产物有促进作用;而反过来Pichia kudriavzevii MJ14对3株乳酸菌产乳酸均有抑制作用,对产乙酸则有促进作用。【结论】建立了一种固态培养方法,结合清香型白酒发酵温度变化规律,有效模拟了实际发酵环境。Pichia kudriavzevii MJ14在与乳酸菌共培养中受到的抑制较小并能有效抑制乳酸菌产乳酸,Saccharomyces cerevisiae YJ1能代谢产生多种风味物质,对清香型白酒酿造有重要意义。     

L-乳酸调控乳酸产生菌产物光学纯度的分析

发酵初期在米根霉菌发酵培养基中添加L-乳酸可以调控发酵产物乳酸的光学纯度。随着L-乳酸添加量的增加,所产L-乳酸的光学纯度随之增加,当L-乳酸的添加量≥1.5g/L时,D-乳酸不再产生。同时,L-乳酸的产量、生物量、糖转化率也随之降低。该调控方法对乳酸菌调控产L-乳酸光学纯度影响不大,对大肠杆菌发酵调控产D-乳酸光学纯度没有效果。     

硫酸锌(ZnSO4)对米根霉发酵产乳酸的影响

[目的]为了了解无机盐与米根霉L-乳酸代谢之间的关系,提高米根霉菌株RLC41-6发酵产L-乳酸的产率与质量,研究了ZnSO4浓度与菌株乳酸代谢和细胞内乳酸脱氢酶活性的关系.[方法]在米根霉培养基中加入不同浓度ZnSO4,经过36℃培养36 h后,应用HPLC-反相色谱法测定产物中的L-乳酸含量,并利用活性PAGE分析法测定细胞内乳酸脱氢酶的活性和组成.[结果]实验结果显示,ZnSO4对除LDH1之外的其它几条同工酶都有促进作用,尤其对LDH4,LDH5作用明显,当ZnSO4浓度大于0.02％时,LDH4,LDH5达到最大水平,同时高浓度的锌离子在体外抑制了LDH的活性.当ZnSO4浓度为0.02％时LDH酶活达到最大200 U/mL,HPLC图谱表明,此时发酵产物的只有L-乳酸,且产量达到最大137g/L,乳酸转化率为91％.[结论]Zn+会影响米根霉的乳酸代谢过程,并导致发酵过程中产物类型的变化,合适浓度的ZnSO4在米根霉代谢产乳酸的过程中,提高了乳酸脱氢酶LDH的表达,抑制丙酮酸进入苹果酸和富马酸途径,从而有利于提高葡萄糖到乳酸的代谢.     

利用五碳糖产高纯度L-乳酸的大肠杆菌基因工程菌的构建

[目的]本研究以已敲除多个产杂酸酶基因的大肠杆菌(Escherichia coli)乙醇工程菌SZ470(△frdBC △ldhA △ackA △focA-pflB △pdhR::pflBp6-pflBrbs-aceEF-lpd)为起始菌株,进一步敲除其乙醇脱氢酶(alcohol dehydrogenase,ADH)基因,同时插入带有自身启动子的乳酸片球菌(Pediococcus acidilactici)的L-乳酸脱氢酶(L-lactate dehydrogenase,LLDH)基因,构建可利用五碳糖同型发酵L-乳酸重组大肠杆菌.[方法]利用λ噬菌体Red重组系统构建乙醇脱氢酶基因(adhE)缺失菌株Escherichia coli JH01,并克隆P.acidilactici的ldhL基因,利用染色体插入技术将其整合到JH01基因组,构建产L-乳酸大肠杆菌基因工程菌Escherichia coli JH12,利用无氧发酵15 L发酵罐测定重组菌株L-乳酸产量.[结果]工程菌JH12在15 L发酵罐中以6％的葡萄糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为1.46 g/(L·h),乳酸生产强度为1.14 g/(L·h),乳酸的产量达到41.13 g/L.发酵产物中未检测到琥珀酸、甲酸的生成,仅有少量乙酸生成,L-乳酸纯度达95.69％(L-乳酸在总发酵产物的比率).工程菌JH12以6％的木糖为碳源进行发酵,发酵到36 h的过程中葡萄糖的消耗速率为0.88 g/(L·h),乳酸生产强度为0.60 g/(L·h),乳酸的产量达到34.73 g/L.发酵产物中杂酸少,乳酸的纯度高达98％.[结论]本研究通过基因敲除、染色体插入及无氧进化筛选获得一株产L-乳酸的大肠杆菌工程菌JH12,该菌株不需利用外源质粒,稳定性好,可利用五碳糖进行发酵,发酵产物中杂酸少,L-乳酸的纯度高.本研究为L-乳酸大肠杆菌工程菌的构建提供一定的技术支持,同时也为大肠杆菌L-乳酸的工业化生产提供了参考依据.     

Novel Method of Lactic Acid Production by Electrodialysis Fermentation

In lactic acid fermentation by Lactobacillus delbrueckii, the produced lactic acid affected the lactic acid productivity. Therefore, for the purpose of alleviating this inhibitory effect, an electrodialysis fermentation method which can continuously remove produced lactic acid from the fermentation broth was applied to this fermentation process. As a result, the continuation of fermentation activity was obtained, and the productivity was three times higher than in non-pH-controlled fermentation. In electrodialysis fermentation, the amount of produced lactic acid was 82.2 g/liter, which was about 5.5 times greater than that produced in non-pH-controlled fermentation. It was concluded that these good results were obtained on account of alleviating the lactic acid inhibitory effect by electrodialysis fermentation. However, the fouling of anion-exchange membranes by cells was observed in electrodialysis fermentation.     

Recovery of lactic acid from fermentation broth by the two-stage process of nanofiltration and water-splitting electrodialysis

A two-stage process of nanofiltration and water-splitting electrodialysis was investigated for lactic acid recovery from fermentation broth. In this process, sodium lactate is isolated from fermentation broth in the first stage of nanofiltration by using an NTR-729HF membrane, and then is converted to lactic acid in the second stage by water-splitting electrodialysis. To determine the optimal operating conditions for nanofiltration, the effects of pressure, lactate concentration, pH and known added impurities were studied. Lactate rejection was less than 5%, magnesium rejection approximated 45%, and calcium rejection was at 40%. In subsequent water-splitting electrodialysis, both the sodium lactate conversion to lactic acid and sodium hydroxide recovery, were about 95%, with a power requirement of 0.9∼1.0 kWh per kg of lactate.     

Acetic Acid Production by an Electrodialysis Fermentation Method with a Computerized Control System

In acetic acid fermentation by Acetobacter aceti, the acetic acid produced inhibits the production of acetic acid by this microorganism. To alleviate this inhibitory effect, we developed an electrodialysis fermentation method such that acetic acid is continuously removed from the broth. The fermentation unit has a computerized system for the control of the pH and the concentration of ethanol in the fermentation broth. The electrodialysis fermentation system resulted in improved cell growth and higher productivity over an extended period; the productivity exceeded that from non-pH-controlled fermentation. During electrodialysis fermentation in our system, 97.6 g of acetic acid was produced from 86.0 g of ethanol; the amount of acetic acid was about 2.4 times greater than that produced by non-pH-controlled fermentation (40.1 g of acetic acid produced from 33.8 g of ethanol). Maximum productivity of electrodialysis fermentation in our system was 2.13 g/h, a rate which was 1.35 times higher than that of non-pH-controlled fermentation (1.58 g/h).     

Integrated bioprocess for the simultaneous production of lactic acid and dairy sewage treatment

An integrated biological process was developed for the conversion of whey lactose to lactic acid. We report about the achievement of maximum COD reduction and thus a substantial unburdening of the environment, combined with the economic production of lactic acid, appropriate for industrial scale. The process – designed for continuous operation – consists of four main steps: (i) Protein recovery by ultrafiltration leading to the first product: protein concentrate. The resulting filtrate is the fermentation substrate acid whey permeate. (ii) Adjustment of the composition of the permeate in the medium preparation step in order to ensure the proper function of the following process steps. (iii) Conversion of the lactose to lactate by fermentation with lactic acid bacteria in a cell recycle reactor, using ceramic microfiltration membranes. (iiii) Conversion of the lactate in the cell-free permeate stream of the fermentation to free lactic acid by bipolar electrodialysis. A stable operation of the process was attained up to more than 2000?hours. Using a new selected strain of lactic acid bacteria, a lactic acid productivity of 17?g?l^?1?h^?1 is achieved at total lactose conversion without any nitrogen supplements like yeast extract. A lactic acid concentration of 190?g?l^?1 is obtained in the acidic cell of the electrodialysis unit and the COD of the remaining sewage is diminished by 92%. As an additional cost reduction item, the neutralization agent of the fermentation is recovered in the caustic cell of the bipolar electrodialysis unit. A cost evaluation for an industrial scale process (100?000?t of whey per year) resulted in a price of 0.66 $ per kg of lactic acid, which under present terms hits the goal of making this process economic for the large scale production of lactic acid as an attractive building block for various purposes in chemical industry.     

Application of bipolar electrodialysis on recovery of free lactic acid after simultaneous saccharification and fermentation of cassava starch

The efficiency of bipolar electrodialysis (BED) for the recovery of lactic acid from fermentation broth was evaluated. Three systems of BED (bipolar-anion, bipolar-cation and bipolar-anion-cation) at fixed voltage (20 V) were compared using a model solution of ammonium lactate (100 g l(-1)). Results showed that bipolar-anion (BED-anion) was the most beneficial in terms of lactate flux, current efficiency, energy consumption and recovery ratio. Consequently, BED-anion was used to purify lactic acid from fermentation broth which had been pre-treated with mono-polar electrodialysis (MED). The final lactic acid concentration and lactate flux obtained were 144 g l(-1) and 393 g m(-2) h(-1), respectively. Using the two-step process (MED and BED-anion) the concentration of fermentation broth was increased by 33% and the total energy consumption was 2.76 kW h kg(-1).     

Isolation of free lactic acid using electrodialysis

Summary Large scale electrodialysis was used to isolate and purify either sodium lactate or free lactic acid from the fermentation broth. In the best cases a four fold concentration was achieved. To obtain a product of a high purity, decolourization followed by a double exchange reaction is recommended.     

Application of metabolically engineered Saccharomyces cerevisiae to extractive lactic acid fermentation

In this study, Saccharomyces cerevisiae OC-2T T165R, metabolically engineered to produce optically pure L(+)-lactic acid, was used to develop a high performance extractive fermentation process. Since the transgenic yeast could produce lactic acid efficiently even at lower than pH 3.5, high extractive efficiency was achieved when tri-n-decylamine (TDA), a tertiary amine, was used as the extractant. Separation of microorganisms by means of a hollow fiber module could not only improve the total amount of lactic acid produced but also increase the lactic acid concentration in the solvent. Moreover, pH had a significant effect on extractive fermentation. The highest rate of recovery of lactic acid could be obtained on pH-uncontrolled fermentation (pH 2.5); however, the lowest amount of lactic acid was produced. Taking into account the trade-off between the fermentation and extraction efficiencies, the optimum pH value was considered to be 3.5, with which the largest amount of lactic acid was produced and the highest lactic acid concentration in the solvent was obtained. The results show promise for the use of the transgenic yeast for extractive fermentation.     

Technological and economic potential of poly(lactic acid) and lactic acid derivatives

Abstract: Lactic acid has been an intermediate-volume specialty chemical (world production ∼ 40,000 tons/yr) used in a wide range of food processing and industrial applications. Lactic acid has the potential of becoming a very large volume, commodity-chemical intermediate produced from renewable carbohydrates for use as feedstocks for biodegradable polymers, oxygenated chemicals, plant growth regulators, environmentally friendly 'green' solvents, and specialty chemical intermediates. The recent announcements of new development-scale plants for producing lactic acid and polymer intermediates by major U.S. companies, such as Cargill, Ecochem (DuPont/ConAgra), and Archer Daniels Midland, attest to this potential.
In the past, efficient and economical technologies for the recovery and purification of lactic acid from crude fermentation broths and the conversion of lactic acid to the chemical or polymer intermediates had been the key technology impediments and main process cost centers. The development and deployment of novel separations technologies, such as electrodialysis (ED) with bipolar membranes, extractive distillations integrated with fermentation, and chemical conversion, can enable low-cost production with continuous processes in large-scale operations. The use of bipolar ED can virtually eliminate the salt or gypsum waste produced in the current lactic acid processes. Thus, the emerging technologies can use environmentally sound processes to produce environmentally useful products from lactic acid. The process economics of some of these processes and products can also be quite attractive. In this paper, the recent technical advances in lactic and polyactic acid processes are discussed. The economic potential and manufacturing cost estimates of several products and process options are presented. The technical accomplishments at Argonne National Laboratory (ANL) and the future directions of this program at ANL are discussed.     

Recovery of carboxylic acids produced by fermentation

Carboxylic acids such as citric, lactic, succinic and itaconic acids are useful products and are obtained on large scale by fermentation. This review describes the options for recovering these and other fermentative carboxylic acids. After cell removal, often a primary recovery step is performed, using liquid–liquid extraction, adsorption, precipitation or conventional electrodialysis. If the carboxylate is formed rather than the carboxylic acid, the recovery process involves a step for removing the cation of the formed carboxylate. Then, bipolar electrodialysis and thermal methods for salt splitting can prevent that waste inorganic salts are co-produced. Final carboxylic acid purification requires either distillation or crystallization, usually involving evaporation of water.     

Formation of aminium lactates in lactic acid fermentation preparation and characterization of 1,4-piperazinium-(L,L)-dilactate obtained from L(+)-lactic acid (part I)

The potential for the production of 1,4-piperazinium-(L, L)-dilactate from L(+)-lactic acid preparations obtained by fermentation was studied. Piperazinium dilactate was found to be a very suitable source material for poly(lactic acid) production. In a novel polymerization process, the intermediate dilactide was directly formed in the salt melt at a moderate temperature. High-performance cultivation of Lactobacillus paracasei on a glucose-MRS medium was carried out using high-viability inocula. After the cell mass had been removed from the fermentation broth by centrifugation and/or ultrafiltration, the lactic acid solution was concentrated to 45% [w/w] by a two-stage electrodialysis process. Two methods of preparing 1,4-piperazinium dilactate were developed: the first from the medium-concentrated lactic acid (45%) and the second from a highly-concentrated lactic acid (85%) obtained by evaporation from the first one. Because there were no physical data on 1,4-piperazinium-(L, L)-dilactate in specialized literature, the pure product was characterized according to its solubility characteristics, melting point and spectroscopic analysis.