Cellulose production from glucose using a glucose dehydrogenase gene (gdh)-deficient mutant of Gluconacetobacter xylinus and its use for bioconversion of sweet potato pulp

A gene fragment encoding a putative pyrroloquinoline quinone glucose dehydrogenase (PQQ GDH) was cloned from a bacterial cellulose (BC)-forming acetic acid bacterium, Gluconacetobacter xylinus (=Acetobacter xylinum) strain BPR 2001, which was isolated as a high BC producer when using fructose as the carbon source. A GDH-deficient mutant of strain BPR 2001, namely GD-I, was then generated via gene disruption using the cloned gene fragment. Strain GD-I produced no gluconic acid but produced 4.1 g.l(-1) of BC aerobically in medium containing glucose as the carbon source. The ability of strain GD-I to convert glucose to BC was approximately 1.7-fold higher than that of the wild type. Strain GD-I was also able to produce 5.0 g.l(-1) of BC from a saccharified solution, which was derived from sweet potato pulp by enzymatic saccharification. Supplementation of ethanol during aerobic cultivation further increased the concentration of BC produced by strain GD-I to 7.0 g.l(-1). The rate of conversion from glucose to BC under these cultivation conditions was equivalent to that of strain BPR 2001 cultivated with fructose as the carbon source.     

琼脂对Komagataeibacter xylinus自发变异的控制

本文研究Komagataeibacter xylinus静态及动态培养中琼脂对其自发变异和细菌纤维素（Bacterial Cellulose,BC）产量及结构的影响。采用涂布法对变异菌率,称重法对纤维素产量以及傅里叶变换红外光谱法和粘度法分别对BC的结晶度和聚合度进行分析,并采用分光光度法对纤维素酶活力测定。结果显示,静态和动态培养中皆出现变异菌,BC产量分别为0.16和0.09 g/L;而静态培养中添加0.05%琼脂和动态培养中添加0.10%琼脂的培养液中没有分离到变异菌株,BC产量分别提高至0.38和0.34 g/L。0.05%琼脂静态培养液连续转接九次过程中没有分离到变异菌株,0.10%琼脂动态培养液连续转接至第二次时,出现变异菌株。以未添加琼脂静态培养为对照,静态培养中,0.05%琼脂使BC的聚合度和结晶度分别下降4.0%和12.9%,动态培养中,0.10%琼脂使BC的结晶度下降了51.2%。尽管纤维素酶活力不受培养方式和琼脂的影响,但动态条件下,因琼脂显著下降了BC的结晶度,纤维素酶易水解BC,因此,BC的聚合度下降了42.2%。     

Increase of xylitol productivity by cell-recycle fermentation of Candida tropicalis using submerged membrane bioreactor

Candida tropicalis, an osmophilic strain isolated from honeycomb, produced xylitol at a maximal volumetric productivity of 3.5 g l(-1) h(-1) from an initial xylose concentration of 200 g l(-1). Even at a very high xylose concentration, e.g., 350 g l(-1), this strain produced xylitol at a moderate rate of 2.07 g l(-1) h(-1). In a fed-batch fermentation of xylose and glucose, 260 g l(-1) xylose was added, and the xylitol production was 234 g l(-1) for 48 h, corresponding to a rate of 4.88 g l(-1) h(-1). To increase xylitol productivity, cells were recycled in a submerged membrane bioreactor with suction pressure and air sparging. For each recycle round in cell-recycle fermentation, the average concentration of xylitol produced, fermentation time, volumetric productivity, and product yield were 180 g l(-1), 19.5 h, 8.5 g l(-1) h(-1), and 85%, respectively. When cell-recycle fermentation was started with the cell mass concentrated twofold after batch fermentation and performed for 10 recycle rounds, we achieved a very high productivity of 12 g l(-1) h(-1). The productivity and total amount of xylitol in cell-recycle fermentation were 3.4- and 11.0-fold higher than those in batch fermentation, respectively.     

Extractive acetone-butanol-ethanol fermentation using methylated crude palm oil as extractant in batch culture of Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564)

The possibility of employing methylated crude palm oil (CPOE) as an extraction solvent to reduce end-product inhibition and to enhance solvent productivity in acetone-butanol-ethanol (ABE) fermentation was evaluated using oleyl alcohol as the standard butanol extractant. Fermentation was carried out at an initial glucose concentration of 90 g/l. CPOE did not inhibit the growth of the fermentative organism. Without solvent extraction, butanol production ceased after 30 h at a concentration of 15.4 g/l limiting cell growth to 3.98 g/l and glucose consumption to 62%. Applying CPOE as the extraction solvent, about 47% of the total butanol produced was extracted, glucose consumption was increased to 83% and relatively high glucose consumption rates and solvent productivities were obtained. Butanol production increased to 20.9 g/l; total ABE solvents and yield also increased from 21.2 g/l and 38% (in conventional fermentation) to 29.8 g/l and 40.4%, respectively.     

Continuous production of wine vinegar in bubble column reactors of up to 60-litre capacity

This paper describes the development of a continuous acetic acid fermentation process for the production of wine vinegar in bubble column reactors of up to 60 l capacity. To determine appropriate fermentation conditions a study of the influence of residual ethanol concentration, inlet flow rate and aeration was carried out using a 6-l laboratory reactor, white table wine as fermentation medium, a temperature of 30 °C and an air flow rate of 0.125 min^-1 (vvm). The concentration of acetic acid obtained in the continuous wine vinegar production ranged from 91 g/l at 28.6 ml/h to 28 g/l at 154.1 ml/h by increasing the inlet flow rate. As expected, the biomass decreased as well, from 208 mg/l to 106 mg/l. The maximum acetification rate was observed in the range 85-110 ml/h, corresponding to a value of about 1.1 g/l/h. A further increase in the flow rate produced a slight decrease in the acetification rate. Best yields, between 94.5 and 94.7%, were obtained in the flow rate range of 60-75 ml/h. The acetification rate was improved only by about 10% by increasing the aeration from 0.125 to 0.250 min^-1. The continuous wine vinegar production was scaled up from the laboratory fermentor to a 60-l pilot acetator. During the steady state (residential time >6), with an inlet flow rate of 950 ml/h, temperature of 30 °C and aeration of 0.250 min^-1, the following parameters were obtained: acetic acid concentration 72 g/l, overall productivity 1.41 g/l/h and yield 94.2%.     

Effective onion vinegar production by a two-step fermentation system

A two-step fermentation system combining a repeated batch process using a flocculating yeast with a charcoal pellet bioreactor was developed for onion vinegar production. Juice from the red onion R-3, which contained 67.3 g/l total sugar, was smoothly converted to onion alcohol containing 30.6 g/l ethanol by repeated batch operation using the flocculating yeast Saccharomyces cerevisiae strain IR-2. Stable operation was possible and the maximum productivity was about 8.0 g/l/h. A packed bed bioreactor containing charcoal pellets produced from waste mushroom medium was then applied to continuous onion vinegar production from the onion alcohol. Onion vinegar was successfully produced, with a maximum productivity and acetic acid concentration of about 3.3 g/l/h and 37.9 g/l, respectively. The total acetic acid yield calculated from the amount of sugar consumed was 0.86. The two-step system was operated for 50 d and proved to be competitive with other systems in terms of its high productivity, high acetic acid yield, operational stability and low production costs.     

Production of cordycepin by a repeated batch culture of a Cordyceps militaris mutant obtained by proton beam irradiation

Cordycepin (3′-deoxyadenosine) is one of the most versatile metabolites of Cordyceps militaris due to its broad spectrum of biological activity. In our previous study, the C. militaris mutant G81-3, which produces higher levels of cordycepin, was obtained by high-energy proton beam irradiation. In this study, the effects of adenosine on cordycepin production in a surface liquid culture of the mutant and the wild type strains were investigated. For the mutant strain, the optimum dose of adenosine yielded a 30% increase in cordycepin production; the maximum levels of production with adenosine and without adenosine were 8.6 g/l and 6.7 g/l, respectively. In contrast, the increase due to adenosine supplementation for the wild type strain was only 15% (3.1 g/l with adenosine and 2.7 g/l without adenosine). Furthermore, a repeated batch culture, an efficient production method, was carried out to eliminate the relatively long lag phase of the mutant culture. Over four cycles, both the mutant and the wild type strain maintained a production level of more than 85% of that of the initial cycle. As a result, the disadvantage of the mutant was successfully overcome, resulting in a productivity (0.48 g/(l d)) higher than that of the batch culture (0.29 g/(l d)). The productivity for cordycepin obtained in this study is the highest reported value to date, and this method could be applied to large-scale production of cordycepin at industrial levels.     

Fed-batch coculture of Lactobacillus kefiranofaciens with Saccharomyces cerevisiae for effective production of kefiran

In a batch coculture of kefiran-producing lactic acid bacteria Lactobacillus kefiranofaciens and lactate-assimilating yeast Saccharomyces cerevisiae, lactate accumulation in the medium was observed, which inhibited kefiran production. To enhance kefiran productivity by preventing lactate accumulation, we conducted lactose-feeding batch operation with feedforward/feedback control during the coculture, so that the lactate production rate of L. kefiranofaciens was balanced with the lactate consumption rate of S. cerevisiae. The lactate concentration was maintained at less than 6 g l(-1) throughout the fed-batch coculture using a 5 l jar fermentor, although the concentration reached 33 g l(-1) in the batch coculture. Kefiran production was increased to 6.3 g in 102 h in the fed-batch coculture, whereas 4.5 g kefiran was produced in 97 h in the batch coculture. The kefiran yield on lactose basis was increased up to 0.033 g g(-1) in the fed-batch coculture, whereas that in the batch coculture was 0.027 g g(-1).     

响应面法优化苹果渣生产细菌纤维素培养基及产物性能研究

为提高木醋杆菌（Acetobacter xylinum）发酵苹果渣水解液生产细菌纤维素（Bacterial cellulose,BC）的产量,采用响应面法对发酵培养基进行优化,同时利用傅里叶红外光谱（FT-IR）和X-射线衍射（XRD）对发酵产物BC的性能和结构进行比较。单因素及响应面实验结果确定木醋杆菌（Acetobacter xylinum）发酵苹果渣水解液生产BC的最佳培养基配方为:蔗糖38.44 g、蛋白胨10.91 g、硫酸镁0.85 g、黄嘌呤0.87 g、乙醇10 m L、苹果渣水解液1000 m L、p H6.0,在此条件下BC的产量为7.19 g/L,较优化前（5.65 g/L）提高了27.3%。苹果渣水解液发酵产物BC结构性能与基本培养基发酵产物BC基本一致。说明苹果渣能够替代部分发酵原料发酵生产BC,且不影响BC性能。      

Klebsiella pneumoniae连续发酵生产1,3-丙二醇的工艺优化

微生物发酵法生产1,3-丙二醇的常用方式是批式流加发酵,但是发酵时间较长,产物的生产强度较低,而连续发酵生产1,3-丙二醇,通过物料不断流动使发酵达到动态平衡,可有效提高生产强度。该文以已建立的批式发酵动力学模型为基础,对克雷伯氏肺炎杆菌(Klebsiella pneumoniae HR526)连续发酵生产1,3-丙二醇的工艺进行了研究,优化得到了使得产物浓度最大的稀释率参数,并分析比较了不同稀释率条件下的单级和多级连续发酵的实验结果。结果表明,最佳的连续发酵方式是二级连续发酵,总稀释率为0.028 h-1时产物浓度达到68.11 g/L,生产强度达到1.89 g/(L·h),与48 h的批式流加发酵相比,生产强度提高了27.7%。     

Enhancement of retinal production by supplementing the surfactant Span 80 using metabolically engineered Escherichia coli

The optimal temperature and pH for retinal production using metabolically engineered Escherichia coli in a 7-l fermentor were found to be 30°C and 7.0, respectively. The agitation speed was a critical factor for retinal production. The optimal agitation speed was 400 rpm (oxygen transfer coefficient, k(L)a, = 92 1/h) in batch culture and 600 rpm (k(L)a=148 1/h) in a fed-batch culture of glycerol. Span 80 was selected as a surfactant for retinal production in metabolically engineered E. coli because Span 80 had proven the most effective for increased retinal production among the tested surfactants. Under the optimal conditions in the fed-batch culture with 5 g/l Span 80, the cell mass and the concentration, content, and productivity of retinal were 24.7 g/l, 600 mg/l, 24.3mg/g-cells, and 18 mg l(-1)h(-1) after 33 h, respectively. They were 1.2-, 2.7-, 2.3-, and 2.7-fold higher than those in the fed-batch culture without Span 80, respectively. The concentration and productivity of retinal in this study were the highest ever reported. The hydrophilic portion of Span 80 (sorbitan) did not affect cell growth and retinal production, but the hydrophobic portion (oleic acid) stimulated cell growth. However, oleic acid plus sorbitan did not stimulate retinal production. Thus, Span 80, as a linked compound of oleic acid and sorbitan produced by esterification, proved to be an effective surfactant for the enhancement of retinal production.     

醋酸菌发酵产细菌纤维素的过程优化

汉逊氏葡糖酸醋杆菌(Gluconacetobacter hansenii)利用传统Hestrin-Scharmm (HS)培养基发酵生产细菌纤维素(bacterial cellulose,BC)的过程中,普遍存在着BC产量不高、葡萄糖利用率低等问题。本研究首先比较了传统HS培养基和改良HS培养基发酵生产BC的结果,改良HS培养基中BC干重产量达到3.34g/L,较传统HS培养基提高了28%,但培养基废液中仍含有41%和70%的残糖和残氮;继而对改良HS培养基一次发酵废液进行优化,添加2.5 g/L酵母粉和1.8 g/L磷酸氢二钠,调节p H至5.9进行二次发酵,可获得3.16 g/L的BC干重,同时发酵液中副产物乙酸浓度仅为一次发酵的一半。综上,利用改良HS培养基发酵结合优化发酵废液进行二次发酵,共获得6.50 g/L的BC干重,是优化前的2.5倍以上,并且葡萄糖的利用率和转化率也分别由56.74%,22.86%提高至88.02%,36.87%。     

Design of a fungal bioprocess for vanillin production from vanillic acid at scalable level by Pycnoporus cinnabarinus

The biotechnological process of vanillin production from vanillic acid by Pycnoporus cinnabarinus was scaled-up at the laboratory level. Vanillin production was studied in two types of bioreactors, a mechanically agitated and an air-lift bioreactor. In the mechanically agitated bioreactor where vanillin was produced in greater quantities, oxygen availability was studied during the growth and production phases. A maximal aeration rate (90l/h equivalent to 0.83 volume of air/volume of medium/min or vvm) during the growth phase and a minimal aeration rate (30 l/h equivalent to 0.28 vvm) during the production phase were necessary to increase vanillin production to 1260 mg/l. Vanillic acid bioconversion to vanillin occurred under the conditions of reduced dissolved oxygen concentration, gentle agitation, high carbon dioxide production and low specific growth rate. However, under these conditions, vanillin production was accompanied by a significant amount of methoxyhydroquinone. Vanillin over a concentration of 1000 mg/l was shown to be highly toxic to the growth of P. cinnabarinus on agar medium. The application of selective XAD-2 resin led to a reduction of vanillin concentration in the medium, thus limiting its toxicity towards the fungal biomass as well as the formation of unwanted by-products such as methoxyhydroquinone and allowed the concentration of vanillin produced to reach 1575 mg/l.     

A mini-scale mass production and separation system for secretory heterologous proteins by perfusion culture of recombinant Pichia pastoris using a shaken ceramic membrane flask

The perfusion culture technique using a shaken ceramic membrane flask (SCM flask) was applied to the production of a secretory heterologous protein. A recombinant methylotrophic yeast strain, Pichia pastoris, was cultured aerobically on a reciprocal shaker using an SCM flask. High-level production of human serum albumin (HSA) was attempted by increasing both the cell concentration and the expression level of the recombinant gene. In the two-stage culture method, the cell concentration was first raised to 17 g/l by feeding glycerol, after which the expression of HSA was induced by feeding methanol. However, the concentration of HSA in the effluent filtrate was as low as 0.15 g/l, while the cell concentration continued to increase. In contrast, HSA was effectively produced by feeding methanol from an early stage of the culture. In this case, the HSA concentration reached 0.24 and 0.46 g/l, respectively, using the growth-associated production method without and with aeration into the head space of the SCM flask. The results showed that supplying sufficient oxygen together with the growth-associated induction method are effective for obtaining high-level expression of the methanol-inducible recombinant gene of P. pastoris. An HSA concentration in the filtrate of 1.5 g/l was finally achieved when the cell concentration was increased to 53 g/l by supplying oxygen-enriched gas to the SCM flask. The yield and productivity of HSA reached 2.6-fold and 10-fold those obtained in an ordinary fed-batch culture using a shake flask, and these levels were readily achieved by continuous replenishment of the culture supernatant. The achievements made in this study should contribute to the development of a handy bioreactor system for mini-scale mass production of target proteins with separation at high purity.     

Production of extracellular bifidogenic growth stimulator (BGS) from Propionibacterium shermanii using a bioreactor system with a microfiltration module and an on-line controller for lactic acid concentration

Production of a bifidogenic growth stimulator (BGS) by Propionibacterium freudenreichii subsp. shermanii (Propionibacterium shermanii) using lactic acid as a carbon source was investigated using different cultivation methods. When a continuous bioreactor system with a filtration device was used at a dilution rate of 0.075 h(-1), the average BGS concentration was 2.4 mg/l, which corresponds to a BGS productivity per cultivation time of 1.8 x 10(-1) mg x l(-1) x h(-1). The BGS productivity per cultivation time in continuous cultivation with filtration was 1.9-fold that (9.4 x 10(-2) mg x l(-1).h(-1)) in a conventional batch cultivation. In fed-batch cultivation with feed-back control using an on-line lactic acid controller with a lactic acid biosensor, it was possible to prevent substrate inhibition by maintaining the lactic acid concentration in culture broth low at 3.3 g/l, and an enhanced BGS production (31 mg/l) was successfully attained. The BGS productivity per cultivation time (2.1x10(-1) mg x l(-1) x h(-1)) in the fed-batch cultivation with feed-back control was 2.2-fold that in the conventional batch cultivation. A new bioreactor system was developed by coupling a continuous bioreactor system with a filtration device to an on-line lactic acid controller. Using the new bioreactor system, we produced BGS continuously at a high level of 47 mg/l. The BGS productivities per cultivation time (3.5 mg.l(-1) x h(-1)) and the total volume of medium used (1.7 x 10(-1) mg x l(-1) x h(-1)) obtained in the new bioreactor system were 37-fold and 2.1-fold those in the conventional batch cultivation, respectively. These results described above clearly demonstrate the positive effects of both the continuous filtration for removal of metabolites (propionic and acetic acids) inhibitory to cell growth and feed-back control of lactic acid concentration in the culture broth on BGS production by P. shermanii. This paper is the first report on BGS production by the propionic acid bacterium using lactic acid as a carbon source.     

Optimization of Citric Acid Production from a New Strain and Mutant of Aspergillus niger Using Solid State Fermentation

A new strain of Aspergillus niger isolated from soil and its mutant were used for citric acid production from carob under solid-state fermentation conditions. The parental strain produced 30 g/kg citric acid, while the mutant G4, selected after four rounds of gamma ray irradiation, produced 60 g/kg. Maximum citric acid production was obtained after 7 days of incubation, as the acid production was 34 and 64 g/kg for parental and mutant strains, respectively. The addition of 2% methanol increased citric acid production from the parental strain to 42 and the mutant G4 to 65 g/kg. Trace elements, namely Cu, Fe, and Zn, promoted the production of citric acid as the acid production from the parental strain increased to 46 g/kg and for mutant G4 increased to 73 g/kg after their addition. The optimum spore inoculum concentration for acid production was 10⁷ ml^-1, and the optimum pH was 5 for both parental and mutant strains.     

响应面法优化木醋杆菌发酵酿酒丢糟水解液产细菌纤维素培养基及其产物性能

为提高木醋杆菌（Acetobacter xylinum）发酵酿酒丢糟水解液生产细菌纤维素（bacterial cellulose,BC）的产量,采用响应面法对发酵培养基进行了优化,同时比较了发酵产物BC的性能和结构。通过单因素及响应面试验结果确定木醋杆菌发酵酿酒丢糟水解液生产BC的最佳培养基配方为：蔗糖39.33 g、蛋白胨20.01 g、MgSO4 0.91 g、柠檬酸钠3.45 g、黄嘌呤1.02 g、乙醇10 mL、酒糟水解液1 000 mL、pH 6.0。在此条件下BC的产量为6.27 g/L,较优化前（4.4 g/L）提高了42.5%。利用傅里叶红外光谱、X射线衍射、扫描电子显微镜对发酵产物BC的性能和结构进行了比较,结果表明,酒糟水解液发酵产物BC结构性能与基本培养基发酵产物BC的基本一致,说明酒糟水解液能够替代部分发酵原料发酵生产BC,且不影响BC性能。     

Ethanol production by a new pentose-fermenting yeast strain, Scheffersomyces stipitis UFMG-IMH 43.2, isolated from the Brazilian forest

The ability of a recently isolated Scheffersomyces stipitis strain (UFMG-IMH 43.2) to produce ethanol from xylose was evaluated. For the assays, a hemicellulosic hydrolysate produced by dilute acid hydrolysis of sugarcane bagasse was used as the fermentation medium. Initially, the necessity of adding nutrients (MgSO(4)·7H(2)O, yeast extract and/or urea) to this medium was verified, and the yeast extract supplementation favoured ethanol production by the yeast. Then, in a second stage, assays under different initial xylose and cell concentrations, supplemented or not with yeast extract, were performed. All these three variables showed significant (p < 0.05) influence on ethanol production. The best results (ethanol yield and productivity of 0.19 g/g and 0.13 g/l/h, respectively) were obtained using the hydrolysate containing an initial xylose concentration of 30 g/l, supplemented with 5.0 g/l yeast extract and inoculated with an initial cell concentration of 2.0 g/l. S. stipitis UFMG-IMH 43.2 was demonstrated to be a yeast strain with potential for use in xylose conversion to ethanol. The establishment of the best fermentation conditions was also proved to be of great importance to increasing the product formation by this yeast strain. These findings open up new perspectives for the establishment of a feasible technology for ethanol production from hemicellulosic hydrolysates.     

常压室温等离子体快速诱变筛选高脯氨酸产率突变株

为提高脯氨酸得率,采用新型常压室温等离子体(ARTP)诱变L-脯氨酸生产菌株———嗜醋酸棒杆菌(出发菌株为谷氨酸生产菌,菌拉丁名ATCC-13870),结合48孔板的高通量筛选手段,在致死率为99%的条件下,获得了16株生长速率和脯氨酸产率变化的菌株。发酵实验结果表明,筛选得到的高产脯氨酸突变体D3在发酵48 h,其脯氨酸浓度从原始菌对照组的54.7 g/L提高到65.8 g/L。     

Optimization of Citric Acid Production from a New Strain and Mutant of Aspergillus niger Using Solid State Fermentation

A new strain of Aspergillus niger isolated from soil and its mutant were used for citric acid production from carob under solid-state fermentation conditions. The parental strain produced 30 g/kg citric acid, while the mutant G4, selected after four rounds of gamma ray irradiation, produced 60 g/kg. Maximum citric acid production was obtained after 7 days of incubation, as the acid production was 34 and 64 g/kg for parental and mutant strains, respectively. The addition of 2% methanol increased citric acid production from the parental strain to 42 and the mutant G4 to 65 g/kg. Trace elements, namely Cu, Fe, and Zn, promoted the production of citric acid as the acid production from the parental strain increased to 46 g/kg and for mutant G4 increased to 73 g/kg after their addition. The optimum spore inoculum concentration for acid production was 10⁷ ml^?1, and the optimum pH was 5 for both parental and mutant strains.