Two-stage pH Control Mode in Batch Fermentation of a Novel Bioflocculant from Corynebacterium Glutamicum

The effect of pH of the fermentation medium on cell growth and the production of a novel bioflocculant(named REA-11) by Corynebacterium glutamicum CCTCC M201005 were investigated. The maximum biomass(2.23 g/L) and flocculating activity(142.2 U/mL) were simultaneously obtained at the 14th hour when the pH value of the culture medium was maintained at 7.0 during the whole fermentation process. The production of REA-11 kept on a trend of increase till the later phase of fermentation process, which resulted in the ultimate flocculating activity of the culture broth to enhance to nearly 100 U/mL at pH 6.0. A two-stage pH control mode was adopted in REA-11 production in which the pH value of the culture medium was controlled at 7.0 during the first 14 h, then decreased to 6. 0 that was maintained until the end of the fermentation process. With the two-stage pH control mode, the maximum flocculating activity reached 178. 8 U/mL which was 30% higher than that obtained under the condition of pH 7.0 and the biomass enhanced about 15%. Compared with the fermentation process without pH control, REA-11 production and cell growth via the two-stage pH control mode increased 80% and 25 %, respectively.     

Bioreactor design considerations in the production of high-quality microbial exopolysaccharide

An examination into the effect of bioreactor design on the production of β,1,3-glucan exopolysaccharide (“curdlan”) by selected patent cultures ofAlcaligenes faecalis andAgrobacterium radiobacter revealed that low shear mixing achieved through the replacement of the radial-flow flat-blade impellers that are commonly supplied in “standard” commercial bioreactors, by low shear (high-pumping) axialflow impellers, leads to an increase in thequality of the exopolymer recovered during the stationary-phase of batch fermentations. Whereas “Rushton turbine” impellers were effective in providing high rates of oxygen transfer necessary for high cell density fermentations, the high shear-to-flow ratio characteristic of this design produced a product of inferior quality, but with characteristics very similar to that of the commercially available “curdlan standard.” Curdlan is water insoluble, and consequently, the fermentation broth is of a relative low viscosity compared to other soluble microbial polysaccharides. Whereas curdlan does not constrain mass transfer from gas to liquid, it nevertheless offers a resistance to oxygen transfer from the liquid to the cell by virtue of the layer of insoluble exopolymer surrounding the cell mass, thereby necessitating an unexpectedly high dissolved oxygen concentration for maximal productivity. The requirement for high volumetric oxygen transfer can be met by low shear designs with axial-flow impellers, providing gas dispersion is assisted by the use of sparging devices consisting of microporous materials.

     

Synthesis and characterization of a novel extracellular polysaccharide by Rhodotorula glutinis

The aim of this work was to characterize an exopolysaccharide by Rhodotorula glutinis KCTC 7989 and to investigate the effect of the culture conditions on the production of this polymer. The extracellular polysaccharide (EPS) produced from this strain was a novel acidic heteropolysaccharide composed of neutral sugars (85%) and uronic acid (15%). The neutral sugar composition was identified by gas chromatography as mannose, fucose, glucose, and galactose in a 6.7:0.2:0.1:0.1 ratio. The molecular weight of purified EPS was estimated to be 1.0−3.8×10⁵ Dalton, and the distribution of the molecular weight was very homogeneous (polydispersity index =1.32). The EPS solution showed a characteristic of pseudoplastic non-Newtonian fluid at a concentration >2.0% in distilled water. The maximum EPS production was obtained when the strain was grown on glucose (30 g/L). Ammonium sulfate was the best suitable nitrogen source for EPS production. The highest yield of EPS was obtained at a carbon to nitrogen ratio of 15. The EPS synthesis was activated at the acidic range of pH 3.0–5.0 and increased when the pH of the culture broth decreased naturally to <2.0 during the fermentation. When the yeast was grown on glucose (30 g/L) and ammonium sulfate (2 g/L) at 22°C at an initial pH of 4.0, EPS production was maximized (4.0 g/L), and the glucose-based production yield coefficient and carbon-based production yield coefficient were 0.30 g of EPS/g of glucose and 0.34 g (carbon of EPS)/g (carbon of glucose), respectively.     

高效液相色谱法同时测定发酵液中赤藓糖醇和L-赤藓酮糖的含量

建立了采用高效液相色谱(HPLC)同时测定发酵液中底物赤藓糖醇和产物L-赤藓酮糖含量的方法。采用Lichrospher 5-NH2色谱柱(250 mm×4.6 mm),柱温30 ℃,以乙腈-水(体积比为9:1)为流动相,流速1.0 mL/min。用示差折光检测器检测赤藓糖醇,检测器温度为35 ℃。用紫外检测器在室温下检测L-赤藓酮糖,检测波长为277 nm。所得赤藓糖醇的线性范围为1.00～100.00 g/L,相关系数为0.9985,检出限为0.10 g/L,定量限为0.45 g/L;所得L-赤藓酮糖的线性范围为1.00～100.00 g/L,相关系数为0.9958,检出限为0.50 g/L,定量限为0.87 g/L;赤藓糖醇的日内和日间相对标准偏差(RSD)分别小于3.28%和5.30%, L-赤藓酮糖的日内和日间RSD分别小于2.16%和2.25%;回收率均大于99%。取不同时间的发酵液样品分别用上述方法测定,结果表明所建立的HPLC法不受发酵液中其他组分的影响,可同时测定底物赤藓糖醇和产物L-赤藓酮糖的含量。     

Acetone–Butanol–Ethanol Production from Waste Seaweed Collected from Gwangalli Beach,Busan, Korea,Based on pH-Controlled and Sequential Fermentation Using Two Strains

The optimal conditions for acetone–butanol–ethanol (ABE) production were evaluated using waste seaweed from Gwangalli Beach, Busan, Korea. The waste seaweed had a fiber and carbohydrate, content of 48.34%; these are the main resources for ABE production. The optimal conditions for obtaining monosaccharides based on hyper thermal (HT) acid hydrolysis of waste seaweed were slurry contents of 8%, sulfuric acid concentration of 138 mM, and treatment time of 10 min. Enzymatic saccharification was performed using 16 unit/mL Viscozyme L, which showed the highest affinity (K_m?=?1.81 g/L). After pretreatment, 34.0 g/L monosaccharides were obtained. ABE fermentation was performed with single and sequential fermentation of Clostridium acetobutylicum and Clostridium tyrobutyricum; this was controlled for pH. A maximum ABE concentration of 12.5 g/L with Y_ABE 0.37 was achieved using sequential fermentation with C. tyrobutyricum and C. acetobutylicum. Efficient ABE production from waste seaweed performed using pH-controlled culture broth and sequential cell culture.     

Study on methane fermentation and production of vitamin B12 from alcohol waste slurry

We studied biogas fermentation from alcohol waste fluid to evaluate the anaerobic digestion process and the production of vitamin B₁₂ as a byproduct. Anaerobic digestion using acclimated methanogens was performed using the continuously stirred tank reactor (CSTR) and fixed-bed reactor packed with rock wool as carrier material at 55°C. We also studied the effects of metal ions added to the culture broth on methane and vitamin B₁₂ formation. Vitamin B₁₂ production was 2.92 mg/L in the broth of the fixed-bed reactor, twice that of the CSTR. The optimum concentrations of trace metal ions added to the culture liquid for methane and vitamin B₁₂ production were 1.0 and 8 mL/L for the CSTR and fixed-bed reactor, respectively. Furthermore, an effective method for extracting and purifying vitamin B₁₂ from digested fluid was developed.     

Xanthan gum production from cassava bagasse hydrolysate with Xanthomonas campestris using alternative sources of nitrogen

Cassava bagasse was hydrolyzed using HCl and the hydrolysate was used for the production of xanthan gum using a bacterial culture of Xanthomonas campestris. Cassava bagasse hydrolysate with an initial concentration of approx 20 g of glucose/L proved to be the best substrate concentration for xanthan gum production. Among the organic and inorganic nitrogen sources tested to supplement the medium—urea, yeast extract, peptone, potassium nitrate, and ammonium sulfate—potassium nitrate was most suitable. Ammonium sulfate was the least effective for xanthan gum production, and it affected sugar utilization by the bacterial culture. In media with an initial sugar concentration of 48.6 and 40.4 g/L, at the end of fermentation about 30 g/L of sugars was unused. Maximum xanthan gum (about 14 g/L) was produced when fermentation was carried out with a medium containing 19.8 g/L of initial reducing sugars supplemented with potassium nitrate and fermented for 72 h, and it remained almost the same until the end of fermentation (i.e., 96 h).     

Value-Added Production of Nisin from Soy Whey

The objective of this study was to evaluate the potential of low/negative value soy whey (SW) as an alternative, inexpensive fermentation substrate to culture Lactococcus lactis subsp. lactis for nisin production. Initially, a microtiter plate assay using a Bioscreen C Microbiology Plate Reader was used for rapid optimization of culture conditions. Various treatments were examined in efforts to optimize nisin production from SW, including different methods for SW sterilization, ultrasonication of soy flake slurries for possible nutrient release, comparison of diluted and undiluted SW, and supplementation of SW with nutrients. In subsequent flask-based experiments, dry bacterial mass and nisin yields obtained from SW were 2.18 g/L and 619 mg/L, respectively, as compared to 2.17 g/L and 672 mg/L from a complex medium, de Man–Rogosa–Sharpe broth. Ultrasonication of soybean flake slurries (10% solid content) in water prior to production of SW resulted in ∼2% increase in biomass yields and ∼1% decrease in nisin yields. Nutrient supplementation to SW resulted in ∼3% and ∼7% increase in cell and nisin yields, respectively. This proof-of-concept study demonstrates the potential for use of a low/negative value liquid waste stream from soybean processing for production of a high-value fermentation end product.     

Effect of environmental factors and carbohydrate on gellan gum production

Submerged culture fermentation studies were carried out in batch mode for optimizing the environmental parameters and carbon source requirement by Pseudomonas elodea for the production of gellan gum. The maximum production of gellan gum was obtained with 16-h-old culture and 8% inoculum at 30°C and pH 7.0 after 52 h of incubation (6.0 g/L). Of the various carbon sources tested, 2% sucrose, glucose, and soluble starch yielded considerably high amounts of gellan. Studies on the concentration of various carbohydrates on gellan gum production indicated that the optimum concentration of glucose and starch was 3%, whereas for sucrose it was 4%. The addition of glucose in the medium above 3% had a detrimental effect on gellan yield. The investigation of intermediate two-step addition of glucose under identical conditions of fermentation showed an enhanced production of gellan (8.12 g/L) as compared with the control (6.0 g/L). To optimize the recovery of gellan from fermented broth, different solvents were tested for precipitation of gellan gum. Among the various solvents tested, tetrahydrofuran gave better recovery of gellan (82%) as compared with the conventional solvent isopropanol (49%).     

高效液相色谱法同时测定生物质乳酸发酵液中有机酸及糖类化合物

建立了高效液相色谱(HPLC)同时测定生物质乳酸发酵液中有机酸及糖类的分析方法。使用Bio-Rad Aminex HPX-87H色谱柱,以5 mmol/L的H2SO4为流动相,在柱温55 ℃,流速0.6 mL/min条件下,采用示差折光检测器进行检测。结果表明,该方法可在17 min内实现发酵液中各种有机酸和糖类化合物等的完全分离与定量,6种有机酸和3种糖类化合物在0.15～5.19 g/L范围内的线性关系良好,回归方程的线性相关系数在0.9998以上。将该法用于米根霉发酵液的检测,两个水平的加标回收率为96.91%～103.11%,相对标准偏差(n=6)为0.81%～4.61%。该法适用于微生物发酵液中多种有机酸和糖类的快速、高效分离和定量测定。     

Role of pyruvate and ascorbate production in regulation of antioxidant enzymes and membrane LPO levels in Fusarium Acuminatum

The role of pyruvate and ascorbate in the regulation of superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase enzymes; and, therefore, membrane lipid peroxidation (LPO) levels in Fusarium acuminatum was investigated in media containing either glycerin or glucose as a carbon source, depending on the incubation period, in the range of 5–25 g/L. Increasing SOD activity between d 9 and 16 of the incubation period showed a positive correlation with a significant increase in pyruvate production up to 15 g/L of glycerin and glucose. In addition, maximum ascorbate production was observed at 15 g/L of glycerin as 82.5 ± 2.1 and 20 g/L of glucose as 54±1.51, whereas CAT activity decreased with an increased concentration of both carbon sources. When compared with the LPO levels determined in media supplemented with glycerin and glucose, the minimum LPO level was 1.88±0.028 nmol of malondialdehyde/g wet wt at 15 g/L of glycerin on d 16, at which it was also observed to have a maximum pyruvate and ascorbate production and SOD, CAT, and GSH-Px activities of 75±1.42 μg/mL, 82.5±2.1 μg/mL, 32.5±0.634 μg/mL, 86.8±2.58 IU/mg, and 1.867 IU/mg, respectively. These results indicate that the biosynthesis of pyruvate and ascorbate may be involved in the regulation of antioxidant enzymes, depending on the glycerin and glucose concentrations, and also this defense network was effective in preventing membrane damage from oxidative stress.     

Biotransformation of uridine monophosphate (UMP) and glucose to uridine diphosphate-glucose (UDPG) byCandida saitoana KCTC7249 cells

The present study investigates the biotransformation of glucose with uridine monophosphate (UMP) to obtain sugar nucleotide, UDP-glucose (UDPG), by the dried cells ofCandida saitoana KCTC7249. The biotransformation was optimized by varying the concentrations of substrates and phosphate ion. UDPG (24 mM) was biotransformed from 200 mM glucose and 37.5 mM UMP by dried cells of C.saitoana. The glucose yields about 64% UDP-glucose, based on UMP concentration. The addition of glucose-1-phosphate to the reaction mixture accelerated the formation of UDPG from a concentration of UMP. The structure of UDP-glucose obtained was determined with¹³C NMR and FAB mass spectra. These results indicate that the yeast-dried cells could be used for the production of nucleotide sugars for donor molecules of complex carbohydrate synthesis.     

Culture medium optimization for acetic acid production by a persimmon vinegar-derived bacterium

A new acetic acid-producing microorganism, Acetobacter sp. RKY4, was isolated from Korean traditional persimmon vinegar, and we optimized the culture medium for acetic acid production from ethanol using the newly isolated Acetobacter sp. RKY4. The optimized culture medium for acetic acid production using this microorganism was found to be 40 g/L ethanol, 10 g/L glycerol, 10 g/L corn steep liquor, 0.5 g/L MgSO₄·7H₂O, and 1.0 g/L (NH₄H₂PO₄. Acetobacter sp. RKY4 produced 47.1 g/L of acetic acid after 48 h of fermentation in a 250 mL Erlenmeyer flask containing 50 mL of the optimized medium.     

Effects of Brewers’ condensed solubles (bcs) on the production of ethanol from low-grade starch materials

Yeast fermentation was performed on grain and bakery byproducts with and without adding the same volume of brewers’ condensed solubles (BCS). Starch material in the grain and bakery byproducts effectively was converted to fermentable sugars with conversion ratios of 93–97% by successive treatments of samples with bacterial αamylase and fungal glucoamylase. The yeast fermentation of these enzyme-digested byproducts alone showed that ethanol concentrations of 16.4–42.7 mL/100 g dry solid in the broth were achieved with fermentation efficiencies of 87–96%. Addition of BCS to the grain byproducts increased ethanol concentration by 10–86% by increasing the potential glucose content of the broth. The rates of fermentation measured by CO₂ gas production demonstrated that BCS addition to bakery byproducts reduced the fermentation time from 62–72 h to 34–35 h. In bakery byproducts that were low in amino nitrogen, exhaustion of nitrogenous compounds in substrates was found to be a limiting factor for yeast growth. Because BCS is a rich source of nitrogen, adding BCS to these substrates markedly increased the fermentation rate.     

Production of biosurfactant by Pseudomonas aeruginosa grown on cashew apple juice

In this work, the ability of biosurfactant production by Pseudomonas aeruginosa in batch cultivation using cashew apple juice (CAJ) and mineral media was evaluated. P. aeruginosa was cultivated in CAJ, which was supplemented with peptone (5.0 g/L) and nutritive broth. All fermentation assays were performed in Erlenmeyer flasks containing 300 mL, incubated at 30 degrees C and 150 rpm. Cell growth (biomass and cell density), pH, and superficial tension were monitored vs time. Surface tension was reduced by 10.58 and 41% when P. aeruginosa was cultivated in nutrient broth and CAJ supplemented with peptone, respectively. These results indicated that CAJ is an adequate medium for growth and biosurfactant production. Best results of biosurfactant production were obtained when CAJ was supplemented with peptone.     

Nomofungin: a new microfilament disrupting agent.

A new alkaloid, nomofungin, has been isolated from the fermentation broth of an unidentified endophytic fungus obtained from the bark of Ficus microcarpa L. The structure of nomofungin was determined by application of spectroscopic methods. The absolute stereochemistry of nomofungin was assigned by using the exciton chirality method. Nomofungin disrupts microfilaments in cultured mammalian cells and is moderately cytotoxic with minimum inhibitory concentrations (MICs) of 2 and 4.5 microg/mL against LoVo and KB cells, respectively. The ring system of nomofungin is unprecedented.     

Production of fumaric acid using rice bran and subsequent conversion to succinic acid through a two-step process

The fungal production of fumaric acid using rice bran and subsequent bacterial conversion of succinic acid using fungal culture broth were investigated. Since the rice bran contains abundant proteins, amino acids, vitamins, and minerals, it is suitable material that fungi use as a nitrogen source. The effective concentration of rice bran to produce fumaric acid was 5 g/L. A large amount of rice bran caused excessive fungal growth rather than enhance fumaric acid production. In addition, we could produce fumaric acid without the addition of zinc and iron. Fungal culture broth containing appro × 25 g/L of fumaric acid was directly employed for succinic acid conversion. The amount of glycerol and yeast extract required for succinic acid conversion was reduced to 70 and 30%, respectively, compared with the amounts cited in previous studies.     

A statistical approach using L<Subscript>25</Subscript> orthogonal array method to study fermentative production of clavulanic acid by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis> MTCC 1142

Clavulanic acid is a naturally occurring antibiotic produced by Streptomyces clavuligerus. The present work reports on clavulanic acid production by Streptomyces clavuligerus MTCC 1142 using one-factor-at-a-time and L₂₅ orthogonal array. The one-factor-at-a-time method was adopted to investigate the effect of media components (i.e., carbon source, nitrogen source and inoculum concentration) and environmental factors such as pH for clavulanic acid production. Production of clavulanic acid by Streptomyces clavuligerus was investigated using seven different carbon sources (viz. glucose, sucrose, modified starch, rice-bran oil, soybean oil, palm oil, and glycerol) and six different nitrogen sources (viz. peptone, yeast extract, ammonium chloride, ammonium carbonate, sodium nitrate and potassium nitrate). A maximum yield of 140 μg/mL clavulanic acid was obtained in the medium containing soybean oil as a carbon source and yeast extract as nitrogen source. Subsequently, the concentration of soybean flour, soybean oil, dextrin, yeast extract and K₂HPO₄ were optimized using L₂₅ orthogonal array method. The final optimized medium produced 500 μg/mL clavulanic acid at the end of 96 h as compared to 140 μg/mL before optimization. Synthesis of precursor molecules as a metabolic driving force is of considerable importance in antibiotic synthesis. Attempts to increase the clavulanic acid synthesis by manipulating the anaplerotic flux on C₃ and C₅ precursors by supplementing the medium with arginine, ornithine, proline, valine, leucine, isoleucine, pyruvic acid and á-ketoglutarate were successful. Supplementing the optimized medium with 0.1 M arginine and 0.1 M leucine increased the yield of clavulanic acid further to 1100 μg/mL and 1384 μg/mL respectively.     

Production of ω-3 polyunsaturated fatty acids from cull potato using an algae culture process

Algal cultivation for converting cull potato to docosahexaenoic acid (DHA) was studied. Schizochytrium limacinum SR21 was selected as the better producing strain, compared with Thraustochytrium aureum because of higher cell density and DHA content. Used as both carbon and nitrogen source, an optimal ratio of hydrolyzed potato broth in the culture medium was determined as 50%, with which the highest production of 21.7 g/L dry algae biomass and 5.35 g/L DHA was obtained, with extra glucose supplemented. Repeat culture further improved the cell density but not fed batch culture, suggesting limited growth was most likely caused by metabolites inhibition.     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.