Production of mannitol by Lactobacillus intermedius NRRL B-3693 in fed-batch and continuous cell-recycle fermentations

Improved fermentation processes were developed for the production of mannitol by a heterofermentative lactic acid bacterium (Lactobacillus intermedius NRRL B-3693). A fed-batch fermentation protocol overcame limitations caused by high substrate concentrations. The process was developed using corn steep liquor and glucose as inexpensive industrial nutrient sources, supplemented with a small amount of soy peptone and manganese. The fed-batch process resulted in a concentration of 176 ± 0.5 g mannitol from 184 ± 0 g fructose and 92 ± 0.1 g glucose per L of final fermentation broth in 30 h with a volumetric productivity of 5.9 g/(L h). Further increases in volumetric productivity of mannitol were obtained in a continuous cell-recycle fermentation process that reached more than 40 g/(L h), despite reduced mannitol levels of 78–98 g/L and residual substrate of 10–20 g/L. This is the first report of such a high volumetric productivity of mannitol by a heterofermentative lactic acid bacterium.     

Effect of salt nutrients on mannitol production by Lactobacillus intermedius NRRL B-3693

The effects of four salt nutrients (ammonium citrate, sodium phosphate, magnesium sulfate, and manganese sulfate) on the production of mannitol by Lactobacillus intermedius NRRL B-3693 in a simplified medium containing 300 g fructose, 5 g soy peptone, and 50 g corn steep liquor per liter in pH-controlled fermentation at 5.0 at 37°C were evaluated using a fractional factorial design. Only manganese sulfate was found to be essential for mannitol production. Added manganese sulfate concentration of 0.033 g/l was found to support maximum production. The bacterium produced 200.6±0.2 g mannitol, 61.9±0.1 g lactic acid, and 40.4±0.3 g acetic acid from 300 g fructose per liter in 67 h.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Effects of pH and corn steep liquor variability on mannitol production by Lactobacillus intermedius NRRL B-3693

Lactobacillus intermedius NRRL B-3693 produced mannitol, lactic acid, and acetic acid when grown on fructose at 37°C. The optimal pH for mannitol production from fructose by the heterofermentative lactic acid bacterium (LAB) in pH-controlled fermentation was at pH 5.0. It produced 160.7 ± 1.1 g mannitol in 40 h with a volumetric productivity of 4.0 g l⁻¹ h⁻¹ in a simplified medium containing 250 g fructose, 50 g corn steep liquor (CSL), and 33 mg MnSO₄ per liter. However, the mannitol production by the LAB was severely affected by the variability of CSL. The supplementation of CSL with soy peptone (5 g/l), tryptophan (50 mg/l), tryptophan (50 mg/l) plus tyrosine (50 mg/l), or commercial protease preparation (2 ml/100 g of CSL) enhanced the performance of the inferior CSL and thus helped to overcome the nutrient limitations.     

A cost effective fermentative production of succinic acid from cane molasses and corn steep liquor by Escherichia coli

AIM: Development and optimization of an efficient and inexpensive medium for succinic acid production by Escherichia coli under anaerobic conditions. METHODS AND RESULTS: Initially, 0.8 gl(-1) of succinic acid was produced in 60 h in 300-ml medium. On optimization, glucose and peptone were replaced by cane molasses and corn steep liquor. Three hundred ml of this medium was inoculated with 4% (v/v) of seed inoculum, incubated at 39 degrees C for 72 h, resulted in 7.1 gl(-1) of succinic acid in 36 h. Scale up in a 10-l fermentor under conditions of controlled pH and continuous CO2 supply in this medium resulted in 17 gl(-1) of succinic acid in 30 h. CONCLUSIONS: A ninefold increase in succinic acid production was obtained in 500-ml anaerobic bottles with optimized medium having cane molasses and corn steep liquor as against initial medium containing glucose and peptone. However, a subsequent scale up in a 10-l fermentor resulted in a 2.5-fold increase in succinic acid production as against optimized medium used in 500-ml anaerobic bottles. SIGNIFICANCE AND IMPACT OF THE STUDY: Succinic acid production was enhanced in medium consisting of inexpensive carbon and nitrogen sources in a shorter span of time.     

Production of poly-β-hydroxybutyrate by Azotobacter vinelandii UWD in media containing sugars and complex nitrogen sources

Summary Vigorously aerated batch cultures of Azotobacter vinelandii UWD formed < 1 g poly--hydroxybutyrate (PHB)/l in media containing pure sugars and 3 g PHB/l in media containing cane molasses, corn syrup or malt extract. However, > 7 g PHB/l was formed when the medium contained 5% beet molasses. Increased yields of PHB were promoted in the media containing pure or unrefined sugars by the addition of complex nitrogen sources. The greatest effect was obtained with 0.05–0.2% fish peptone (FP), proteose peptone no. 3 or yeast extract. Peptones caused a 1.6-fold increase in residual non-PHB biomass and up to a 25-fold increase in PHB content. Hence the increased PHB formation was not simply due to stimulation of culture growth. The amount of PHB per cell protein formed by UWD in media containing FP was greatest in glucose = corn syrup > malt extract > sucrose = fructose = cane molasses > maltose, as carbon sources. The addition of FP to medium containing beet molasses did not stimulate PHB yield. The peptone effect was most significant in well-aerated cultures, which were fixed nitrogen and consuming glucose at a high rate. An explanation for the peptone effect on PHB yield stimulation is proposed.     

Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: effect of various carbon and nitrogen sources

Recombinant Escherichia coli (ATCC:PTA-1579) harbouring poly(3-hydroxybutyrate) (PHB) synthesising genes from Streptomyces aureofaciens NRRL 2209 accumulates PHB. Effects of different carbon and nitrogen sources on PHB accumulation by recombinant E. coli were studied. Among the carbon sources used glycerol, glucose, palm oil and ethanol supported PHB accumulation. No PHB accumulated in recombinant cells when sucrose or molasses were used as carbon source. Yeast extract, peptone, a combination of yeast extract and peptone, and corn steep liquor were used as nitrogen sources. The maximum PHB accumulation (60% of cell dry weight) was measured after 48 h of cell growth at 37 degrees C in a medium with glycerol as the sole carbon source, and yeast extract and peptone as nitrogen sources. Scanning electron microscopy of the PHB granules isolated from recombinant E. coli revealed these to be spherical in shape with a diameter ranging from 0.11 to 0.35 pm with the mean value of 0.23 +/- 0.06 pm.     

碳源和氮源对5-酮基-葡萄糖酸生成的影响

氧化葡萄糖杆菌Gluconobacter oxydans可以将葡萄糖氧化成葡萄糖酸,并进一步氧化成2-酮基-葡萄糖酸(2KGA)和5-酮基-葡萄糖酸(5KGA),其中5KGA在催化剂的作用下能够转化为L(+)-酒石酸。为了提高5-酮基-葡萄糖酸产量,以仅生成5KGA的氧化葡萄糖杆菌Gluconobacter oxydans HGI-1为出发菌株,研究不同碳源(蔗糖、乳糖、麦芽糖、淀粉、葡萄糖)和有机氮源(酵母浸粉、鱼粉、玉米浆、黄豆饼粉、棉籽饼粉)对5KGA产量的影响。500 mL摇瓶试验结果表明,当葡萄糖浓度为100 g/L时,5KGA产量最高为98.20 g/L;当有机氮源为酵母浸粉、鱼粉和玉米浆,其添加量的蛋白含量为1.60%时,5KGA产量分别为100.20 g/L、109.10 g/L和99.83 g/L,其中,使用鱼粉的5KGA产量最高,使用玉米浆的5KGA产量比酵母浸粉略低。出于经济考虑,文中选择玉米浆作有机氮源,并在5 L发酵罐中进行分批发酵放大试验,5KGA的产量为93.80 g/L,最大生成速率为3.48 g/(L·h),平均生成速率为1.56 g/(L·h)。结果表明,葡萄糖和玉米浆分别为Gluconobacter oxydans HGI-1规模化生产5KGA的最适碳源和氮源,可利用葡萄糖几乎全部(85.93%)转化为5KGA。     

Use of a sequential strategy of experimental design to optimize the inulinase production in a batch bioreactor

A potential application of inulinase in the food industry is the production of fructoligosaccharides (FOS) by the transfructosilation of sucrose. The FOSs present many interesting functional properties besides their ability to increase the shelf-life and flavor of many products. The use of an industrial medium represents a good alternative to producing inulinase at low cost, since the activity may improve, or at least remain the same, as that obtained using a synthetic medium. This work was an optimization study of the inulinase production by Kluyveromyces marxianus NRRL Y-7571 using industrial pre-treated culture medium in a bioreactor employing a sequential strategy of experimental design. Initially, a Plackett–Burman (Screening Design) design was used, where the studied variables were molasses, corn steep liquor, yeast extract concentration, and agitation and aeration rates. After the analysis of the effects, a central composite rotational design (CCRD) was carried out. The optimized condition for the inulinase production was: 250 g/l of molasses, 80 g/l of corn steep liquor, 6 g/l of yeast extract, 300 rpm of agitation and 1.5 vvm aeration rate, which resulted in an enzymatic activity of 1,317 ± 65 U/ml.     

Production of a novel polygalacturonic acid bioflocculant REA-11 by Corynebacterium glutamicum

The production of a novel polygalacturonic acid bioflocculant REA-11 from a newly isolated strain, Corynebacterium glutamicum CCTCC M201005, was investigated. Sucrose was chosen as a carbon source for REA-11 production. Complex nitrogen sources containing urea and an organic nitrogen compound enhanced both bacterial growth and REA-11 production, among which urea plus corn steep liquor was shown to be the most efficient combination. A cost-effective medium for REA-11 production mainly comprised 17 g/l sucrose, 0.45 g/l urea, and 5 ml/l corn steep liquor, under which conditions the flocculating activity reached 390 U/ml. The molar ratio of carbon to nitrogen (C/N) significantly affected REA-11 production, where a C/N ratio of 20:1 was shown to be the best. Interestingly, by simultaneously feeding sucrose and urea at a C/N ratio of 20:1 at 24 h of fermentation, REA-11 production (458 U/ml) was enhanced by 17% compared to the control. In a 10 l jar fermentor, lower dissolved oxygen tension was favorable for REA-11 production: a flocculating activity of 520 U/ml was achieved at a kappaLa of 100 h(-1). REA-11 raw product is relatively thermo-stable at acidic pH ranges of 3.0-6.5. Preliminary application studies showed that REA-11 had stronger flocculating activity to Kaolin clay suspension compared to chemical flocculants. In addition, the capability of decolorizing molasses wastewater indicates the industrial potential of this novel bioflocculant.     

High-level production of heterologous proteins using untreated cane molasses and corn steep liquor in Escherichia coli medium

To develop an economical industrial medium, untreated cane molasses (UCM) was tested as a carbon source for fermentation culturing of Escherichia coli. To test the industrial application of this medium, we chose a strain co-expressing a carbonyl reductase (PsCR) and a glucose dehydrogenase (BmGDH). Although corn steep liquor (CSL) could be used as an inexpensive nitrogen source to replace peptone, yeast extract could not be replaced in E. coli media. In a volume of 40 ml per 1-l flask, a cell concentration of optical density (OD₆₀₀) 15.1 and enzyme activities of 6.51 U/ml PsCR and 3.32 U/ml BmGDH were obtained in an optimized medium containing 25.66 g/l yeast extract, 3.88 g/l UCM, and 7.1% (v/v) CSL. When 3.88 g/l UCM was added to the medium at 6 h in a fed-batch process, the E. coli concentration increased to OD₆₀₀ of 24, and expression of both PsCR and BmGDH were twofold higher than that of a batch process. Recombinant cells from batch or fed-batch cultures were assayed for recombinant enzyme activity by testing the reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (S)-4-chloro-3-hydroxybutanoate (CHBE). Compared to cells from batch cultures, fed-batch cultured cells showed higher recombinant enzyme expression, producing 560 mM CHBE in the organic phase with a molar yield of 92% and an optical purity of the (S)-isomer of >99% enantiomeric excess.     

Factors affecting lipase production in Syncephalastrum racemosum

Syncephalastrum racemosum grown as a static culture showed maximum lipase production at 30°C in 2d at pH 8.0. When the medium was supplemented with fructose, maximum production of lipase per unit of growth was achieved, followed by raffinose, sucrose, ribose, galactose, maltose, lactose, mannitol and glucose. Amongst the nitrogen sources tested, corn steep liquor at 8% (v/v) produced maximum enzyme; there was evidence of catabolite repression by glucose when groundnut protein, soybean meal, milk casein or wheat bran were the sources of nitrogen. Calcium, potassium and sodium citrates, each at 0.1% (w/v), increased the yield of lipase.     

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。     

Production of glucose oxidase using Aspergillus niger and corn steep liquor

Glucose oxidase production was optimized using an isolated strain of Aspergillus niger and an economical nutrient source, corn steep liquor (CSL). The culture produced 580 +/- 30 units/ml of the enzyme using 70 g/l sucrose as the carbon source. Using CSL as the sole nutrient source enzyme synthesis was increased to 640 +/- 36 units/ml. None of the nitrogen sources (nitrates of calcium, sodium, ammonium, potassium and yeast extract, malt extract, and peptone) was beneficial to the enzyme synthesis. Aeration and agitation enhanced enzyme synthesis to 850 +/- 45 units/ml. Glucose oxidase has numerous applications in food industry and clinical fields.     

The utilization of beet molasses as a novel carbon source for cephalosporin C production by Acremonium chrysogenum: Optimization of process parameters through statistical experimental designs

In this work, cephalosporin C (CPC) production on pilot scale fermenters of 600l capacity with 350l working volume by Acremonium chrysogenum EMCC 904 was performed. The effects of fermentation medium composition, inoculum concentration, initial pH and aeration rate on CPC production by A. chrysogenum strain was investigated by using response surface methodology (RSM). The Plackett-Burman design which involves two concentrations of each nutrient was effective in searching for the major medium components promoting CPC production. Under our experimental conditions; Soya oil, beet molasses and corn steep liquor were found to be the major factors contributing to the antibiotic production. Subsequently, a Box-Behnken design was used for outlining the concentration of the most effective medium constituents. Estimated optimum composition for the production of CPC was as follows: soya oil, 40g/l; beet molasses, 180g/l; and corn steep liquor, 330g/l. The central composite design was used for outlining the optimum values of the fermentation parameters. Estimated optimum values for the production of CPC are as follows: inoculum level, 10(5.5)spores/ml; initial pH, 4.3; and aeration rate, 9364ml/min.     

水产动物益生菌乳酸杆菌LH发酵工艺的研究

目的从生产实际出发,对1株高效乳酸杆菌（Lactobacillus spp）LH进行液体发酵培养基优化及发酵条件研究。方法通过碳源、氮源、无机盐、促生长素等单因子筛选及正交试验设计获得以下最佳培养基：糖蜜12 g/L,酵母膏5 g/L,蛋白胨1 g/L,葡萄糖4 g/L,玉米浆3 g/L,乙酸钠5 g/L,NaC l 5 g/L,K2HPO42.5 g/L,KH2PO42.5 g/L,MgSO40.5g/L,MnSO40.25 g/L。在此培养基上研究了该菌株最佳发酵条件。结果培养基初始pH 6.0,接种量2%（v/v,相对装液量）,500 m l三角瓶中装液量为500 m l,发酵温度为30～35℃,静置培养。在最佳培养条件下,LH活菌量达到1.74×10^9CFU/m l。结论通过活菌平板计数法测定了乳酸杆菌LH生长曲线,24 h为最佳种龄,生产收获时间是36 h。     

Effects of Corn Steep Liquor and Thiamine on l-Glutamic Acid Fermentation of Hydrocarbons: IV. Utilization of Hydrocarbons by Microorganisms

The effect of nutrients of natural source, such as corn steep liquor, peptone, and yeast extract, on the fermentative production of L-glutamic acid from hydrocarbons by a Corynebacterium was studied. Corn steep liquor and meat extract were found to be remarkably stimulatory to L-glutamic acid production; about 5 g per liter of L-glutamic acid were accumulated in a culture broth containing 3% n-paraffins, 0.01% corn steep liquor, and mineral salts. Among nutritional factors contained in corn steep liquor, biotin had very little effect on the accumulation of L-glutamic acid, but thiamine was highly stimulatory to L-glutamic acid production. The optimal concentration of thiamine for L-glutamic acid production was 3 to 5 μg per liter, and for cell growth, 50 μg per liter. L-Glutamic acid was accumulated in negligible quantity when the amount of thiamine in the culture broth was sufficient to support abundant growth of bacterial cells.     

响应面法优化毛霉菌发酵培养基

采用响应面分析方法优化毛霉菌B的发酵培养基,首先通过单因素试验筛选出葡萄糖为最适碳源,酵母膏和玉米浆为最适氮源,用Plackett—Burman试验对葡萄糖、酵母膏、玉米浆、MgSO4、FeSO4、NILCl/、HPO4进行评估并筛选出具有显著效应的3个因素：葡萄糖、酵母膏、玉米浆,再通过最陡爬坡试验逼近其最大响应区域,最后采用Box—Behnken试验对其用量进行优化,得到毛霉菌最佳发酵培养基（g/L）：葡萄糖51．54,酵母膏5．22,玉米浆14．31,MgSO40．5,FeSO40．1,NH4Cl3,k2HPO43,pH6．0～6．5。培养基优化后,毛霉生物量由23．51g／L提高至31．13g/L,比对照组提高32．41％,腺嘌呤转化率由53．59％提高至59．97％,ATP产率由6．56g／L提高至7．34g／L,比对照组提高11．89％。     

Citric acid production by a novel Aspergillus niger isolate: II. Optimization of process parameters through statistical experimental designs

In this work, sequential optimization strategy, based on statistical designs, was employed to enhance the production of citric acid in submerged culture. For screening of fermentation medium composition significantly influencing citric acid production, the two-level Plackett-Burman design was used. Under our experimental conditions, beet molasses and corn steep liquor were found to be the major factors of the acid production. A near optimum medium formulation was obtained using this method with increased citric acid yield by five-folds. Response surface methodology (RSM) was adopted to acquire the best process conditions. In this respect, the three-level Box-Behnken design was applied. A polynomial model was created to correlate the relationship between the three variables (beet molasses, corn steep liquor and inoculum concentration) and citric acid yield. Estimated optimum composition for the production of citric acid is as follows pretreated beet molasses, 240.1g/l; corn steep liquor, 10.5g/l; and spores concentration, 10(8)spores/ml. The optimum citric acid yield was 87.81% which is 14 times than the basal medium. The five level central composite design was used for outlining the optimum values of the fermentation factors initial pH, aeration rate and temperature on citric acid production. Estimated optimum values for the production of citric acid are as follows initial pH 4.0; aeration rate, 6500ml/min and fermentation temperature, 31.5 degrees C.     

Fed-batch fermentation with and without on-line extraction for propionic and acetic acid production byPropionibacterium acidipropionici

Fed-batch propionic and acetic acid fermentations were performed in semi-defined laboratory medium and in corn steep liquor withPropionibacterium acidipropionici strain P9. On average, over four experiments, 34.5 g/l propionic acid and 12.8 g/l acetic acid were obtained in about 146 h in laboratory medium with 79 g/l glucose added over five feeding periods. The highest concentration of propionic acid, 45 g/l, was obtained when the glucose concentration was not allowed to drop to zero. In corn steep liquor 35 g/l propionic acid and 11 g/l acetic acid were produced in 108 h from 59.4 g/l total lactic acid provided as seven feedings of corn steep liquor. Extractive fed-batch fermentations were conducted in semi-defined medium using either flat-sheet-supported liquid membranes or hollow-fiber membrane extraction to remove organic acids from the culture medium. As operated during the course of the fermentation, these systems extracted 25% and 22% of the acetic acid and 36.5% and 44.5% of the propionic acid, respectively, produced in the fermentation. Total amounts of acids produced were about the same as in comparable nonextractive fermentations: 30–37 g/l propionic acid and 13 g/l acetic acid were produced in 150 h. Limitations on acid production can be attributed to limited substrate feed, not to failure of the extraction system.Journal paper J-16303 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project 3122.     

Growth Kinetics of Clostridium bifermentans and Its Ability to Degrade TNT Using an Inexpensive Alternative Medium

A strain of Clostridium bifermentans isolated from a munitions-supplemented anaerobic digester is known to degrade 2,4,6-trinitrotoluene (TNT) in rich media such as Brain Heart Infusion (BHI) broth. In order to make this biodegradation process commercially feasible, a new growth medium was developed. Corn steep liquor and molasses were selected as possible nitrogen and carbon sources. A medium containing 2.4% corn steep liquor and 0.4% molasses was chosen based on the value for maximum specific growth rate. The values of µ_m and K_s from continuous runs were of 0.029 min^-1 and 0.488 g/L, respectively. To reduce the overhead cost for maintaining an anoxic environment for this obligatory anaerobic bacterium, the threshold oxygen level under which the bacterium can survive was determined. The degradation of TNT was then carried out in a batch bioreactor, and in a continuously stirred tank bioreactor. The solubility of TNT was enhanced by using the surfactant, Tween 80, and the optimal concentration of Tween 80 was determined to be 2.5%. This concentration of Tween 80 increased TNT solubility by 100% while the effect on growth rate was minimal. Degradation of TNT was then carried out in a continuously stirred tank bioreactor and was found to follow Michaelis-Menten-type kinetics. Degradation of TNT was found to be possible under limited oxygen presence, but at a reduced degradation rate.