Actinobacillus succinogenes 130Z厌氧发酵产丁二酸条件初步研究

对Actinobacillus succinogenes130Z厌氧发酵产丁二酸的培养条件进行了初步研究。研究了不同有机氮源,不同碳、氮源浓度配比、CO2供体、培养温度,培养基起始pH值对菌株生长和产酸的影响,并在5 L发酵罐中进行了放大试验。结果表明最佳培养基配方为(g/L):葡萄糖10,酵母膏5,NaHCO310,Na2HPO40.3,NaH2PO4.2H2O 9.6,K2HPO43,MgCl20.2,MnCl20.2,NaCl 0.1;pH7.0。在最佳条件下,血清瓶37℃培养24 h,丁二酸产量达到8.3 g/L,在5 L发酵罐中培养,葡萄糖质量浓度分别为10和100 g/L时,丁二酸产量分别达到8.2和45.6 g/L,收率分别为80%和65%。

Actinobacillus succinogenes NJ113产丁二酸过程中的底物抑制

研究了分批发酵条件下以葡萄糖作为底物对产琥珀酸放线杆菌Actinobacillus succinogene NJ113发酵产丁二酸的影响,针对底物抑制现象,采用变速补料控制发酵罐中葡萄糖浓度的补料分批发酵方式.结果表明,发酵过程中将葡萄糖浓度控制在0～10g/L,以Na2CO3作为pH调节剂,经26h厌氧发酵,消耗60g/L葡萄糖,能积累45.27g/L丁二酸,得率达75.45%,生产强度为1.74g/(L·h),比初始葡萄糖浓度为60g/L的分批发酵周期缩短了18.75%,主产物丁二酸的得率和生产强度分别提高了5.44%和31.82%,副产物甲酸产量有所减少,而乙酸产量有所增加.通过代谢网络中相关酶的酶活分析,解析了补料过程中主副产物的分布.

Actinobacillus succinogenes NJ113利用乳清厌氧发酵制备丁二酸

考察产琥珀酸放线杆菌(A.succinogene)NJ113利用乳清作为C源厌氧发酵对菌体生长和产丁二酸能力的影响.结果表明:A.succinogenes NJ113能够充分利用乳糖进行发酵合成丁二酸.当乳清质量浓度为70 g/L(以乳糖计)时,丁二酸质量浓度最高达48.44 g/L.以乳清为发酵底物,当酵母膏添加量为6 g/L时,丁二酸产量达到48.47 g/L,与酵母膏添加量为10 g/L时的丁二酸产量基本相当.以干酪乳清作为C源进行发酵制备丁二酸,与葡萄糖发酵结果相比,丁二酸产量为47.5 g/L,质量收率为67.9%,比葡萄糖发酵低9.6%,同时副产物乙酸较多,丁二酸与乙酸的质量比值较低.

有机酸对Actinobacillus succinogenes厌氧发酵过程的抑制作用

通过在发酵培养基中分别添加不同浓度的丁二酸、甲酸和乙酸,考察了3种有机酸对菌体生长及代谢产物积累的影响。结果表明,在初始葡萄糖浓度为40 g/L厌氧发酵产丁二酸体系中,甲酸抑制作用最强,乙酸次之,丁二酸无明显抑制作用。当甲酸和乙酸总浓度超过13.70 g/L时,菌体开始衰亡。利用膜循环生物反应器在发酵过程甲酸和乙酸总浓度达到13.70 g/L时移出部分抑制性代谢产物,有效地降低了有机酸对A.succi-nogenes NJ113生长代谢的抑制作用,丁二酸生产强度达1.70 g/(L.h),比分批发酵提高了17.2%。

Actinobacillus succinogenes NJ113厌氧发酵产丁二酸培养条件的优化

采用Plackett-Burman设计法(Plackett-Burman,PB),对影响Actinobacillus succinogenes NJ113厌氧发酵生产丁二酸的11个因子进行了筛选。结果表明,影响该菌厌氧发酵产丁二酸的主要因子为葡萄糖、酵母膏、玉米浆。在此基础上,采用响应曲面法(Response Surface Methodology,RSM)对这3个因子的影响进行了研究,得出丁二酸产量的数学模型,通过对二次多项回归方程求解,得到3因子的最优用量:葡萄糖107g/L,酵母膏16 g/L,玉米浆12 g/L,在优化条件下培养48 h,丁二酸的产量由原始培养条件下的62g/L增到84 g/L,收率从62%提高到78.5%,生产强度达1.75 g/(L·h)。

Study of Succinic Acid Production by Actinobacillus Succinogenes

Succinic acid has recently emerged as an important chemical （commodity） because it can be used for the manufacturing of synthetic resins and biodegradable polymers and as an intermediate for chemical synthesis. Till date, succinic acid is mainly produced by chemical processes, however, due to the environmental concerns and the concepts of sustainability, researches are directed towards the production of succinic acid by microbial fermentation. The fact that carbon dioxide （CO2） is needed by the microorganisms for succinic acid production is another interesting feature. The fermentation was carried out with Actinobacillus succinogenes using a two-level fractional factorial design 2sl. The variables analyzed and their levels were： concentration of glucose, yeast extract, temperature, pH and agitation. The results show that the variables that more influenced on succinic acid production were pH, temperature and yeast extract.

Optimization of succinic acid fermentation with Actinobacillus succinogenes by response surface methodology(RSM)

Succinic acid is considered as an important platform chemical. Succinic acid fermentation with Actinobacillus succinogenes strain BE-1 was optimized by central composite design （CCD） using a response surface methodology （RSM）. The optimized production of succinic acid was predicted and the interactive effects between glucose, yeast extract, and magnesium carbonate were investigated. As a result, a model for predicting the concentration of succinic acid production was developed. The accuracy of the model was confirmed by the analysis of variance （ANOVA）, and the validity was further proved by verification experiments showing that percentage errors between actual and predicted values varied from 3.02% to 6.38%. In addition, it was observed that the interactive effect between yeast extract and magnesium carbonate was statistically significant. In conclusion, RSM is an effective and useful method for optimizing the medium components and investigating the interactive effects, and can provide valuable information for succinic acid scale-up fermentation using A. succinogenes strain BE-I.

Efect of the Gad system on Actinobacillus succinogenes during acid stress

The glutamate decarboxylase(Gad)system is an important amino acid-dependent acid resistance system commonly found in microorganisms.Actinobacillus succinogenes is one of the best natural producers of succinic acid(SA)but lacks glutamate decarboxylase.This study assessed the efects of Gad system introduction into A.succinogenes.The recombinant strains gadB-SW and gadBC-SW were constructed by heterologous expression of gadB alone,or gadB together with gadC,respectively.After 1.0 and 1.5 h of acid stress at pH 4.6,cell survival of gadBC-SW was greater than gadB-SW.The growth of gadB-SW and gadBC-SW was both afected by the expression of heterologous proteins and byγ-aminobutyric acid,with gadBC-SW growth reduced at a neutral pH.SA production in acidic conditions was evaluated by a shake fask and by 3-L bioreactor fermentation.The results showed gadBC-SW to increase SA production by 8.4%in shake fask compared to the parent strain,SW.For a 3-L bioreactor batch fermentation under acidic environment,the highest conversion rate of sugar to SA was observed for gadBC-SW,reaching 96%.However,SA concentration by gadBC-SW was only 47 g/L and 31 g/L at pH 6.5 and pH 6.0,respectively.In summary,the introduction of heterologous gadB and gadC into A.succinogenes not only improved acid tolerance but also infuenced the synthesis of SA and added a metabolic burden.

利用甘蔗糖蜜半连续发酵生产琥珀酸

In this work, the semi-continuous succinic acid fermentation by Actinobacillus succinogenes CGMCC1593 from cane molasses with a novel two-stage and two-stream system was studied, in order to increase the succinic acid productivity.The fermentation conditions of the first stage, such as initial sugar concentration, medium addition volume, and time interval of each loading were optimized.Under the optimized condition the maximum succinic acid productivity (2.38 g·L-1·h-1) was increased by 111% and 114% compared with those obtained in batch and fed batch fermentation, respectively.In addition, the maximum succinic acid concentration (46.0 g·L-1)increased by 12.9% compared with that in batch fermentation, and reached a compatible level of that obtained in fed batch fermentation (36 h, 48.8 g·L-1).This process was operated stably for 39 circles without any decreases in both succinic acid concentration and productivity.During the 39 operation circles, the fermentation time of each batch (stage 1 and stage 2), the average succinic acid concentration, succinic acid productivity, succinic acid yield, and sugar conversion rate were 21—24 h, 43.5 g·L-1, 2.07 g·L-1·h-1, 0.79 g·g-1, and 96.4%, respectively.

菊芋原料同步糖化发酵生产丁二酸

对菊芋原料发酵生产丁二酸进行了研究,用Actinobacillus succinogenes和Aspergillusniger同步糖化发酵,发现同步糖化发酵效果优于糖化后再发酵,在同步糖化发酵过程中还原糖质量浓度始终保持在10～40 g/L,可以避免高浓度的还原糖对A.succinogenes的抑制。5 L搅拌罐中同步糖化补料分批发酵96 h产丁二酸98.2 g/L,对消耗糖产率95.4%,生产强度1.02g/（L.h）。

利用甘蔗糖蜜厌氧发酵产丁二酸的研究

对甘蔗糖蜜作为廉价碳源厌氧发酵制备丁二酸进行初步研究。实验证明,Actinobacillus succinogenes NJ113能够利用葡萄糖、果糖、蔗糖等碳源,制备丁二酸为菌体利用廉价甘蔗糖蜜作碳源进行丁二酸发酵提供可行性依据。通过比较不同的糖蜜预处理方法,得出经硫酸处理的糖蜜发酵效果最好,丁二酸浓度37.73g/L,比未经处理的糖蜜所产丁二酸浓度高12.6%。考察不同的糖蜜添加量对发酵结果的影响表明,初始总糖浓度为65g/L时,丁二酸的产量最高为49.63g/L;在3L罐中进行放大实验,丁二酸产量46.91g/L,质量收率为72.2%,分别比混合糖(含蔗糖、果糖、葡萄糖)的发酵结果高9.8%、10%。

利用纤维素水解液中的纤维二糖发酵制备丁二酸

纤维素水解液中通常含有纤维二糖。本文考察了Actinobacillus succinogenes NJ113利用纤维二糖厌氧发酵生产丁二酸的能力,并利用蔗渣纤维素制备纤维二糖作为碳源用于厌氧发酵生产丁二酸。3 L发酵罐厌氧发酵结果显示:以35 g/L纤维二糖作为碳源发酵制备丁二酸,其产量为23.51 g/L,产率达到67.17%;用含有18 g/L纤维二糖和17 g/L其它糖类的蔗渣纤维素水解液作为碳源发酵制备丁二酸,丁二酸的产量和产率分别为20.00 g/L和64.73%。因此,Actinobacillus succinogenes NJ113具有较强的利用纤维二糖生产丁二酸的能力,而且利用废弃的纤维素制备纤维二糖作为碳源高效、经济地发酵制备丁二酸具有可行性。

发酵法生产丁二酸的化学合成培养基的筛选

以产琥珀酸放线杆菌Actinobacillus succinogenes NJ113为出发菌株,针对该菌株筛选出含有关键生长因子的化学合成培养基,其关键因子为谷氨酸(Glu)、蛋氨酸(Met)和生物素(V H)和烟酸(V PP)。结合原发酵培养基中的磷酸缓冲盐成分,最终得到的化学合成培养基配方(g/L):CH3COONa 1.36,NaCl 1.0,MgCl20.2,CaCl20.2,Na2HPO40.31,NaH2PO41.6,KH2PO43,NH4HCO31.57,Glu 0.87,Met 0.11,V H0.010,V PP0.025。在3 L发酵罐上进行验证实验,50 g/L初始葡萄糖发酵70 h,丁二酸的质量浓度为45.2 g/L,丁二酸收率达到90.4%。与之前的半合成培养基发酵制备丁二酸相比,丁二酸的收率提高了25.2%,副产物也有很大幅度的减少。

耐铵型产琥珀酸放线杆菌的选育

以Actinobacillus succinogenes NJ113为出发菌株,经硫酸二乙酯(DES)诱变,在含8～20 g/L硫酸铵平板中筛选到一株耐铵型突变株YZ25,该菌株在含8 g/L硫酸铵培养基中厌氧发酵,琥珀酸产量达32.68 g/L,比出发菌提高了180.5%,对葡萄糖收率达65.4%,副产物乙酸、甲酸产量分别下降3.5%、28.7%,琥珀酸/乙酸比值由0.63提高到2.5。在7.5 L发酵罐中,用氨水调节pH分批实验,发酵34 h琥珀酸产量达27.13 g/L,较出发菌株提高了85.3%。

利用甘蔗糖蜜与乳清粉厌氧发酵制备丁二酸

考察了甘蔗糖蜜替代昂贵葡萄糖作为碳源、乳清粉替代大部分酵母粉作为氮源时,对Actinobacillus succinogenes NJ113发酵制备丁二酸的影响。血清瓶厌氧发酵结果证明:对照组(葡萄糖40 g/L)的丁二酸产量仅为26.04 g/L,而以糖蜜为碳源(以总还原糖计算为40 g/L)时,丁二酸产量达到28.27 g/L,比对照组提高了8.57%。在此基础上,以糖蜜为碳源、不同比例的乳清粉和酵母粉为混合氮源发酵制备丁二酸,确定了糖蜜、乳清粉和酵母粉混合使用的最佳浓度分别为40 g/L、8 g/L和2 g/L。此外,在3 L发酵罐体系中添加40 g/L糖蜜、8 g/L乳清粉、2 g/L酵母粉进行发酵试验,实验结果证明:丁二酸终产量达到32.54 g/L,收率达到81.13%。

Enhanced Succinic Acid Production from Sake Lees Hydrolysate by Dilute Sulfuric Acid Pretreatment and Biotin Supplementation

Succinic acid is valued as a potential starting point for the production of chemicals of the C4 family or in the prepara-tion of biodegradable polymers. For sustainable development in this era of petroleum shortage, production of succinic acid by microbial fermentation of renewable feedstock has attracted great interest. In this study, pretreatment with sulfuric acid and biotin supplementation were used to enhance succinic acid production by Actinobacillus succinogenes 130Z from sake lees, a byproduct of Japanese rice wine. Pretreatment with sulfuric acid resulted in little change of glucose, total nitrogen and succinic acid content in the sake lees hydrolysate but had a positive effect on succinic acid fermentation, which caused a 25.0% increase in succinic acid yield in batch fermentation. Biotin supplementation was used to further enhance the fermentability of sake lees hydrolysate. As a result, a 30 h batch fermentation of 0.5% sulfuric acid pretreated sake lees hydrolysate with 0.2 mg/L biotin gave a succinic acid yield of 0.59 g/g from 61.6 g/L of glucose, with a productivity of 1.21 g/(L?h). A 22.9% increase in succinic acid yield and a 101.7% increase in succinic acid productivity were obtained compared with untreated sake lees hydrolysate.

Enhanced direct gaseous CO_(2) fixation into higher bio-succinic acid production and selectivity

Utilizing CO_(2)for bio-succinic acid production is an attractive approach to achieve carbon capture and recycling(CCR)with simultaneous production of a useful platform chemical.Actinobacillus succinogenes and Basfia succiniciproducens were selected and investigated as microbial catalysts.Firstly,the type and concentration of inorganic carbon concentration and glucose concentration were evaluated.6 g C/L MgCO_(3)and 24 g C/L glucose were found to be the optimal basic operational conditions,with succinic acid production and carbon yield of over 30 g/L and over 40%,respectively.Then,for maximum gaseous CO_(2)fixation,carbonate was replaced with CO_(2)at different ratios.The“less carbonate more CO_(2)”condition of the inorganic carbon source was set as carbonate:CO_(2)=1:9(based on the mass of carbon).This condition presented the highest availability of CO_(2)by well-balanced chemical reaction equilibrium and phase equilibrium,showing the best performance with regarding CO_(2)fixation(about 15 mg C/(L·hr)),with suppressed lactic acid accumulation.According to key enzymes analysis,the ratio of phosphoenolpyruvate carboxykinase to lactic dehydrogenase was enhanced at high ratios of gaseous CO_(2),which could promote glucose conversion through the succinic acid path.To further increase gaseous CO_(2)fixation and succinic acid production and selectivity,stepwise CO_(2)addition was evaluated.50%-65%increase in inorganic carbon utilization was obtained coupled with 20%-30%increase in succinic acid selectivity.This was due to the promotion of the succinic acid branch of the glucose metabolism,while suppressing the pyruvate branch,along with the inhibition on the conversion from glucose to lactic acid.

产琥珀酸菌的快速筛选及鉴定

琥珀酸是重要的化工原料,广泛应用于食品、医药等领域。采用简单、高效的筛选方法,从牛瘤胃内容物分离获得1株有潜力的琥珀酸生产菌。通过生理生化分析和16SrDNA同源性比对,鉴定为琥珀酸放线杆菌(Actinobacillus succinogenes)。