产酸丙酸杆菌生长特性及5L罐发酵动力学研究

论文在摇瓶水平对产酸丙酸杆菌基本生长特性（温度、pH、摇床转速、接种量、种龄等）、碳源、氮源利用情况、产物抑制及5 L罐发酵动力学进行了研究。结果表明,该菌在32℃,初始pH 6．5,摇床转速150 r/min,接种24 h的种子液,接种量为5％条件下,产酸丙酸杆菌生长及产酸水平达最高值;该菌可利用碳源十分广泛,但对氮源要求比较高,只可利用有机氮源;在不同初始葡萄糖浓度下,产酸丙酸杆菌生长及产酸水平差异不大,无明显底物抑制现象;在2g/L的初始丙酸盐浓度下,该菌生长受到明显抑制;在5L发酵罐中,初始葡萄糖浓度为58．8 g/L,发酵72 h,葡萄糖消耗完全,丙酸终浓度达22．4 g/L,丙酸得率和产率分别达0．381 g/g和0．295 g/（L·h）,丙酸占总酸比例达72．10％。     

米根霉利用纯糖和不同预处理玉米秸秆酶解糖生产L-乳酸

通过单因素实验设计,优化米根霉摇瓶发酵产L-乳酸。在此基础上,以蒸气爆破和碱处理玉米秸秆酶解液为混合C源,与纯糖对比,研究不同预处理玉米秸秆混合C源对米根霉发酵产L-乳酸的影响。结果显示:在初始葡萄糖质量浓度100g/L、(NH4)2SO4质量浓度2g/L、接种量6%(体积分数)、转速170r/min、发酵12h后添加30g/LCaCO3的条件下,米根霉发酵产L-乳酸质量浓度为69.15g/L。米根霉发酵不同预处理玉米秸秆酶解混合C源,木糖的存在影响了米根霉的C代谢网络,降低L乳酸的产量。     

四环素抗性重组鼠李糖乳杆菌的构建

将含有鼠李糖乳杆菌同源序列的自杀质粒pUC-ldhD-Ter转化到鼠李糖乳杆菌JCM 1553中,成功获得2株含四环素抗性的重组鼠李糖乳杆菌GL-1和GL-2。采用PCR技术鉴定重组菌株染色体基因组上含有四环素抗性基因Ter;生长曲线说明四环素抗性基因的插入对鼠李糖乳杆菌的生长没有较大影响。抗性菌株GL-1和GL-2在含10%葡萄糖的摇瓶发酵培养基发酵结果显示:37℃,200 r/min发酵40h,菌体密度OD₆₀₀最大达到25.49和24.66,残糖含量为0.60%和0.63%,L-乳酸最高产量为93.956 g/L、93.693 g/L,葡萄糖转化率达到94.82%、94.56%,与原始菌株没有显著区别。     

氮源对L-苏氨酸发酵的影响

以L-苏氨酸生产菌TRFC为供试菌株,研究了氮源对L-苏氨酸发酵的产量和糖酸转化率的影响。首先通过摇瓶实验确定发酵的最佳无机氮源和有机氮源分别为硫酸铵和酵母粉,进一步利用10L罐补料分批发酵确定硫酸铵和酵母粉的最佳用量,继续优化培养条件,采用发酵中后期流加硫酸铵和糖氨混合补料等措施,L-苏氨酸产量得到进一步的提高。在最优发酵条件下,通过10L罐补料分批发酵36h,产酸可达118.9g/L,糖酸转化率为47.6%。     

戊糖乳杆菌发酵培养基氮源条件的优化

对戊糖乳杆菌发酵培养基的氮源条件进行了优化。通过单因素实验及响应面分析优化利用木糖高产乳酸的戊糖乳杆菌发酵培养基的不同氮源组合。优化得到的牛肉膏与柠檬酸氢二铵复合的最佳组成为牛肉膏17.72 g/L,柠檬酸氢二铵1.91 g/L,得到乳酸实际最大产量42.37 g/L。添加玉米浆与酵母粉和无机氮源复合的最佳组成为玉米浆46.54 g/L,酵母粉21.95 g/L,柠檬酸氢二铵9.95 g/L,可得到乳酸最大产量41.06 g/L。通过响应面优化减少了有机氮源的种类。牛肉膏与柠檬酸氢二铵的复合得到了更高的乳酸产量,且减少了有机氮源用量,节约了成本。玉米浆与酵母粉的复合解决了单一玉米浆造成的木糖利用速率过低的问题,同样得到较高浓度的乳酸。     

菊糖芽孢乳杆菌利用麸皮水解液发酵生产D-乳酸

考察菊糖芽孢乳杆菌YBS1-5利用麸皮的水解液发酵生产D-乳酸的性能。首先研究了不同蛋白酶对麸皮中蛋白组分的水解效率,优选酸性蛋白酶并对其进行水解工艺的优化,最终其水解液中的含氮量为4.6 g/L,水解效率为85.8%。对酸性蛋白酶的水解液残渣进行稀酸预处理后,利用纤维素酶对其进行酶解。通过批次补料酶解,水解液中的还原糖质量浓度达141.2 g/L,其中葡萄糖质量浓度为138.1 g/L、木糖质量浓度为1.4 g/L。利用麸皮的蛋白酶水解液和纤维素酶水解液替代葡萄糖和酵母粉发酵制备D-乳酸。在96 h内,D-乳酸产量达99.5 g/L,生产速率达1.04 g/(L·h),转化率89.1%。     

采用玉米秸秆水解糖和玉米浆发酵生产丁二酸

研究了以玉米秸秆水解糖为碳源,不同氮源条件下琥珀酸放线杆菌Actinobacillus succinogenesSF-9的丁二酸发酵产酸能力。结果表明玉米浆可以替代酵母膏作为丁二酸发酵的廉价氮源。厌氧摇瓶丁二酸发酵单因素试验,得到在初糖浓度50 g/L时,玉米浆的较佳用量为20 g/L。在5 L搅拌罐上,考察了不同初始玉米秸秆水解糖浓度对A.succinogenes SF-9发酵生产丁二酸的影响,结果显示高初始秸秆糖浓度对琥珀酸放线杆菌的生长有抑制作用。采用补料分批发酵,发酵60 h丁二酸的产量达到42.7g/L,丁二酸产率82.7%,生产强度0.81 g/(L·h)。丁二酸的产量和生产强度较分批发酵有明显提高。     

利用农业废弃物发酵生产D-乳酸

利用农业废弃物玉米芯酶解液替代葡萄糖作为碳源,棉籽粕替代酵母膏作为氮源发酵生产D-乳酸。结果表明:在初始还原糖质量浓度为100 g/L(葡萄糖88.5 g/L,木糖11.5 g/L)、棉籽粕3.5 g/L、每升发酵体积添加3 U的中性蛋白酶以及pH 6.5的情况下,采取补料发酵措施,菌株Sporolactobacillus sp.YBS1-5在90 h内产生了111.8 g/L的D-乳酸,糖酸转化率为87%,光学纯度达98%以上,生产强度达1.24 g/(L·h)。本文提供了一种利用农业废弃物发酵产D-乳酸的新途径。     

产D-乳酸重组大肠杆菌ptsG基因的敲除及其混合糖同步发酵

为构建能够同时高效利用五碳糖和六碳糖发酵产D-乳酸的重组大肠杆菌工程菌,以能高效利用五碳糖发酵产D-乳酸的大肠杆菌工程菌E.coli JH13为出发菌株,通过Red同源重组技术敲除葡萄糖跨膜转运基因pts G。实验结果表明,pts G缺陷菌株E.coli JH15在10%混合糖(5%葡萄糖和5%木糖)培养基中发酵,可同时利用五碳糖和六碳糖以完成发酵;而对照菌葡萄糖消耗完才利用木糖,发酵结束还有18 g/L木糖残留;JH15乳酸产量为83.04 g/L,相比于对照菌株提高了25.86%;在稻草秸秆水解液中发酵,JH15同时利用葡萄糖、木糖和L-阿拉伯糖,乳酸产量为25.15 g/L,转化率为86.42%。JH15作为能利用混合糖同步发酵产D-乳酸的大肠杆菌工程菌,它的成功构建为利用廉价的木质纤维素水解物为原料发酵生产D-乳酸提供参考依据。     

乳杆菌Lactobacillus sp.lxp发酵高产L-乳酸研究

筛选得到一株乳杆菌Laetobaeillus sp．,进行发酵生产高浓度L-乳酸的研究。考察了种龄、接种量、温度和不同pH调节剂对乳酸发酵的影响。结果表明：最佳种子培养时间为15h;最佳接种量为15％;最适培养温度为42℃;与氨水和氢氧化钠相比,碳酸钙更适于作为发酵过程的pH调节刺;以葡萄糖为碳源,添加豆粕水解液和玉米浆作为辅料,2L罐培养120h,L-乳酸质量浓度可达202 g/L,糖转化率91．3％,L-乳酸占发酵液中总酸含量98％以上。     

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.     

发酵产丁二酸过程中废弃细胞的循环利用

对厌氧发酵产丁二酸后的废弃细胞进行破壁处理,考察了以细胞水解液作为有机氮源重新用于丁二酸发酵的可行性。比较了超声破碎、盐溶、酶解3种方法破碎细胞获得的水解液作为氮源发酵产丁二酸的效果,结果表明酶解制得的细胞水解液效果最佳。以总氮含量为1.11g/L的酶解液(相当于10g/L酵母膏)作为氮源发酵,丁二酸产量可达42.0g/L,继续增大酶解液用量对耗糖、产酸能力没有显著提高。将细胞酶解液与5g/L酵母膏联用发酵36h后,丁二酸产量达75.5g/L,且丁二酸生产强度为2.10g/(L·h),比使用10g/L酵母膏时提高了66.7%。因此,厌氧发酵产丁二酸结束后的废弃细胞酶解液可以替代原培养基中50%的酵母膏用于发酵。     

Utilization of Molasses Sugar for Lactic Acid Production by Lactobacillus delbrueckii subsp. delbrueckii Mutant Uc-3 in Batch Fermentation

Efficient lactic acid production from cane sugar molasses by Lactobacillus delbrueckii mutant Uc-3 in batch fermentation process is demonstrated. Lactic acid fermentation using molasses was not significantly affected by yeast extract concentrations. The final lactic acid concentration increased with increases of molasses sugar concentrations up to 190 g/liter. The maximum lactic acid concentration of 166 g/liter was obtained at a molasses sugar concentration of 190 g/liter with a productivity of 4.15 g/liter/h. Such a high concentration of lactic acid with high productivity from molasses has not been reported previously, and hence mutant Uc-3 could be a potential candidate for economical production of lactic acid from molasses at a commercial scale.     

Enhancement of ribitol production during fermentation of <Emphasis Type="Italic">Trichosporonoides oedocephalis</Emphasis> ATCC 16958 by optimizing the medium and altering agitation strategies

To maximize the productivity of ribitol, which is an important starting material for the production of one expensive rare sugar, L-ribose, the effects of culture medium and agitation speed on cell growth as well as on the productivity of ribitol were thoroughly investigated in a 7 L fermentor. The maximum volumetric productivity, 0.322 g/L/h of ribitol, were obtained at an initial glucose concentration of 200 g/L in a batch culture. Based on the optimum glucose concentration, the ribitol yield conversed from glucose was up to 0.193 g/g when 1% yeast extract was used as a nitrogen source. When the agitation speed was maintained at 200 rpm, the ribitol concentration of 38.60 g/L was collected after 120 h of cultivation time. Additionally, the scheme of two-phase agitation and glucose infusion was employed. To begin, in the first 24 h of fermentation, a high agitation rate at 350 rpm and the initial glucose concentration of 50 g/L were applied, and the biomass concentration of 25.50 g/L was achieved at 36 h of incubation; whereas this value was observed until 60 h in the former batch fermentation methods. Then, in the second phase, with the agitation speed reduced to 150 rpm and the infusion amount of glucose controlled at 150 g/L, the yield of ribitol reached to 65.00 g/L in two-phase agitation fermentation and was 1.68 fold of that obtained in one-stage batch fermentation. To our knowledge, this study first demonstrates its significant effectiveness in improving ribitol production with the application of Trichosporonoides oedocephalis ATCC 16958.     

Utilization of by-products derived from bioethanol production process for cost-effective production of lactic acid

The by-products of bioethanol production such as thin stillage (TS) and condensed distillers solubles (CDS) were used as a potential nitrogen source for economical production of lactic acid. The effect of those by-products and their concentrations on lactic acid fermentation were investigated using Lactobacillus paracasei CHB2121. Approximately, 6.7 g/L of yeast extract at a carbon source to nitrogen source ratio of 15 was required to produce 90 g/L of lactic acid in the medium containing 100 g/L of glucose. Batch fermentation of TS medium resulted in 90 g/L of lactic acid after 48 h, and the medium containing 10 % CDS resulted in 95 g/L of lactic acid after 44 h. Therefore, TS and CDS could be considered as potential alternative fermentation medium for the economical production of lactic acid. Furthermore, lactic acid fermentation was performed using only cassava and CDS for commercial production of lactic acid. The volumetric productivity of lactic acid [2.94 g/(L·h)] was 37 % higher than the productivity obtained from the medium with glucose and CDS.     

Production of lactic acid by Lactobacillus rhamnosus with vitamin-supplemented soybean hydrolysate

Batch fermentation studies were performed to evaluate the potentials of a complex nitrogen source, soybean, as an alternative to yeast extract for the economical production of lactic acid by Lactobacillus rhamnosus. An enzyme-hydrolysate of soybean meal, Soytone, with an adequate supplementation of vitamins was found to be highly effective in supporting lactic acid production from glucose and lactose. The effects of seven selected vitamins: d-biotin, pyridoxine, p-aminobenzoic acid, nicotinic acid, thiamine, pantothenic acid, and riboflavin, on cell growth and lactic acid production were investigated to provide the basis for the optimization of vitamin supplementation to minimize the cost. Pantothenic acid was the most required compound while the other six vitamins were also essential for high lactic acid productivity. As a result of the optimization, 15 g/l yeast extract could be successfully replaced with 19.3 g/l Soytone supplemented with the vitamins, resulting in a production of 125 g/l lactic acid from 150 g/l glucose. The volumetric productivity and lactate yield were 2.27 g/l/h and 92%, respectively, which were higher than those with 15 g/l yeast extract. The raw material cost was estimated to be 21.4 cent/kg lactic acid, which was only approximately 41% of that with yeast extract.     

Optimisation of media and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441

Process variables and concentration of carbon in media were optimised for lactic acid production by Lactobacillus casei NRRL B-441. Lactic acid yield was inversely proportional to initial glucose concentration within the experimental area (80-160 g l(-1)). The highest lactic acid concentration in batch fermentation, 118.6 g l(-1), was obtained with 160 g 1(-1) glucose. The maximum volumetric productivity, 4.4 g 1(-1) h(-1) at 15 h, was achieved at an initial glucose concentration of 100 g l(-1). Similar lactic acid concentrations were reached with a fedbatch approach using growing cells, in which case the fermentation time was much shorter. Statistical experimental design and response surface methodology were used for optimising the process variables. The temperature and pH optima for lactic acid production were 35 degrees C, pH 6.3. Malt sprout extract supplemented with yeast extract (4 g l(-1)) appeared to be an economical alternative to yeast extract alone (22 g l(-1)) although the fermentation time was a little longer. The results demonstrated both the separation of the growth and lactic acid production phases and lactic acid production by non-growing cells without any nutrient supplements. Resting L. casei cells converted 120 g l(-1) glucose to lactic acid with 100% yield and a maximum volumetric productivity of 3.5 g l(-1) h(-1).     

Production of lactic acid and byproducts from waste potato starch by <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis>: role of nitrogen sources

Summary Lactic acid was produced by Rhizopus arrhizus using waste potato starch as the substrate. The aim of this study was to identify the role of nitrogen sources and their impact on the formation of lactic acid and associated byproducts. Ammonium sulphate, ammonium nitrate, urea, yeast extract and peptone were assessed in conjunction with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio favoured the production of fumaric acid. Ammonium nitrate appeared to be the most suitable nitrogen source for achieving a high and stable lactic acid yield, and minimizing the production of byproducts such as biomass and ethanol, while urea proved to be the least favourable nitrogen source. Yeast extract and peptone appeared to improve fungal cell growth. The kinetics data revealed that a high concentration of ammonium nitrate enhanced the lactic acid productivity. The maximum lactic acid concentration of 36.4 g/l, representing a yield of 91%, was obtained with addition of 0.909 g/l ammonium nitrate in 32 h.