Strain development and medium optimization for fumaric acid production

Rhizopus oryzae RUR709 mutant was isolated based on halo size from selection medium via mutagenesis with UV and γ-rays, and the production of fumaric acid in the submerged fermentation was assessed. The maximum concentration of fumaric acid was obtained using 0.5% corn steep liquor (CSL) as the nitrogen source. Organic nitrogen sources were shown to be more effective in fumaric acid production than inorganic nitrogen sources. Using optimum medium obtained by response surface methodology (RSM), the maximum concentration of fumaric acid achieved in flask culture was 26.2 g/L, which is fairly close to the 27.4 g/L predicted by the model. The highest concentration of fumaric acid in the stirred-tank reactor generated by the R. oryzae RUR709 mutant was 32.1 g/L and yield (0.45 g/g) and productivity (0.32 g/L/h) were highest at 4 days.     

SSF production of lactic acid from cellulosic biosludges

The use of cellulosic biosludges generated in a Kraft pulp mill was investigated as substrate for lactic acid production by simultaneous saccharification and fermentation (SSF). The effect of the operation mode (batch or fedbatch), the initial liquid to solid ratio (12 or 30 g/g) and the nutrient supplementation (MRS components or none) on several parameters including lactic acid concentration, volumetric productivity and product yields, were evaluated. When the operation was carried out in fedbatch mode with nutrient supplementation and using a LSR(0)=12 g/g, a broth containing 42 g/L was obtained after 48 h with a volumetric productivity of 0.87 g/L h and a product yield of 37.8 g lactic acid/100 g biosludges. In a similar experiment carried out without nutrient supplementation, a lactic acid concentration of 39.4 g/L was obtained after 48 h with a volumetric productivity of 0.82 g/L h and a product yield of 35.5 g L-lactic acid/100 g biosludges.     

Simultaneous Production and Recovery of Fumaric Acid from Immobilized Rhizopus oryzae with a Rotary Biofilm Contactor and an Adsorption Column

An integrated system of simultaneous fermentation-adsorption for the production and recovery of fumaric acid from glucose by Rhizopus oryzae was investigated. The system was constructed such that growing Rhizopus mycelia were self-immobilized on the plastic discs of a rotary biofilm contactor during the nitrogen-rich growth phase. During the nongrowth, production phase, the biofilm was alternately exposed to liquid medium and air upon rotation of the discs in the horizontal fermentation vessel. The product of fermentation, fumaric acid, was removed simultaneously and continuously by a coupled adsorption column, thereby moderating inhibition, enhancing the fermentation rate, and sustaining cell viability. Another beneficial effect of the removal of fumaric acid is release of hydroxyl ions from a polyvinyl pyridine adsorbent into the circulating fermentation broth. This moderates the decrease in pH that would otherwise occur. Polyvinyl pyridine and IRA-900 gave the highest loading for this type of fermentation. This fermentation system is capable of producing fumaric acid with an average yield of 85 g/liter from 100 g of glucose per liter within 20 h under repetitive fed-batch cycles. On a weight yield basis, 91% of the theoretical maximum was obtained with a productivity of 4.25 g/liter/h. This is in contrast to stirred-tank fermentation supplemented with calcium carbonate, whose average weight yield was 65% after 72 h with a productivity of 0.9 g/liter/h. The immobilized reactor was operated repetitively for 2 weeks without loss of biological activity.     

法夫酵母PLX-All发酵纤维素酶水解物合成虾青素

法夫酵母（Phaffia rhodozyma)PLX-All菌株能够发酵纤维素酶水解物进行虾青素的生物合成。纤维素的酶解物主要为纤维二糖和葡萄糖,在另外添加适量其它营养物后可被法夫酵母发酵用于生长及合成虾青素。摇瓶试验结果表明,培养108h,法夫酵母的生物量可达2.3g／L,虾青素的产率达913.4g／g干细胞,虾青素体积产率为2.1mg／L。在2L罐的发酵试验中,法夫酵母的生物量可达3.23g／L（第96h）,虾青素的产率达581.4g／g干细胞,虾青素体积产率达1.88mg／L。     

法夫酵母PLX-All发酵纤维素酶水解物合成虾青素*

法夫酵母（Phaffia rhodozyma)PLX-All菌株能够发酵纤维素酶水解物进行虾青素的生物合成。纤维素的酶解物主要为纤维二糖和葡萄糖,在另外添加适量其它营养物后可被法夫酵母发酵用于生长及合成虾青素。摇瓶试验结果表明,培养108h,法夫酵母的生物量可达2.3g／L,虾青素的产率达913.4g／g干细胞,虾青素体积产率为2.1mg／L。在2L罐的发酵试验中,法夫酵母的生物量可达3.23g／L（第96h）,虾青素的产率达581.4g／g干细胞,虾青素体积产率达1.88mg／L。     

不同pH调节剂对产琥珀酸放线杆菌NJ113发酵产丁二酸的影响

在3L发酵罐中分别采用不同的碱性物质作为pH调节剂,考察其对产琥珀酸放线杆菌Actinobacillus succinogenes NJ113厌氧发酵制备丁二酸的影响。结果表明:Ca2+、NH4+调节剂对菌体生长代谢有较大阻碍作用,丁二酸产量较低;采用含Na+调节剂,在发酵中后期菌体出现絮凝现象严重,且产丁二酸能力骤降;采用含Mg2+调节剂,整个发酵过程菌体代谢旺盛,发酵效果较佳。根据各碱性物质的调节能力以及对菌体生长代谢的影响,选择NaOH、Mg(OH)2和Na2CO3、Mg(OH)2分别作为混合碱组分调节pH,并对两组混合碱中各物质的质量比例进行优化。结果表明,以NaOH、Mg(OH)2混合,两者质量比为1:1时,发酵效果最好,丁二酸质量浓度高达到69.8g/L,质量收率74.5%。该种混合碱配比可有效替代碱式MgCO3调节pH,既达到高产丁二酸的目的,又可降低生物制备丁二酸的成本。     

Optimization of L-malic acid production by Aspergillus flavus in a stirred fermentor

Effects of various nutritional and environmental factors on the accumulation of organic acids (mainly L-malic acid) by the filamentous fungus Aspergillus flavus were studied in a 16-L stirred fermentor. Improvement of the molar yield (moles acid produced per moles glucose consumed) of L-malic acid was obtained mainly by increasing the agitation rate (to 350 rpm) and the Fe(z+) ion concentration (to 12 mg/L) and by lowering the nitrogen (to 271 mg/L) and phosphate concentrations (to 1.5 mM) in the medium. These changes resulted in molar yields for L-malic acid and total C(4) acids (L-malic, succinic, and fumaric acids) of 128 and 155%, respectively. The high molar yields obtained (above 100%) are additional evidence for the operation of part of the reductive branch of the tricarboxylic acid cycle in L-malic acid accumulation by A. flavus. The fermentation conditions developed using the above mentioned factors and 9% CaCO(3) in the medium resulted in a high concentration (113 g/L L-malic acid from 120 g/L glucose utilized) and a high overall productivity (0.59 g/L h) of L-malic acid. These changes in acid accumulation coincide with increases in the activities of NAD(+)-malate dehydrogenase, fumarase, and citrate synthase.     

Genome-shuffling improved acid tolerance and L-lactic acid volumetric productivity in Lactobacillus rhamnosus

Genome shuffling is an efficient approach for the rapid improvement of industrially important microbial phenotypes. Here we improved the acid tolerance and volumetric productivity of an industrial strain Lactobacillus rhamnosus ATCC 11443 by genome shuffling. Five strains with subtle improvements in pH tolerance and volumetric productivity were obtained from the populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis, and then they were subjected for recursive protoplast fusion. A library that was more likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both ultraviolet irradiation and heat treatments. After three rounds of genome shuffling, four strains that could grow at pH 3.6 were obtained. We observed 3.1- and 2.6-fold increases in lactic acid production and cell growth of the best performing at pH 3.8, respectively. The maximum volumetric productivity was 5.77+/-0.05 g/lh when fermented with 10% glucose under neutralizing condition with CaCO(3), which was 26.5+/-1.5% higher than the wild type.     

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).     

出芽短梗霉产聚苹果酸发酵条件的优化

【背景】出芽短梗霉可发酵葡萄糖生成聚苹果酸,但存在转化率和转化效率低等瓶颈,阻碍其实现商业化生产。【目的】通过优化发酵培养条件,提高出芽短梗霉的聚苹果酸产量、糖酸转化率和生产强度。【方法】采用单因素试验优化适宜出芽短梗霉BK-10菌株产生聚苹果酸的培养条件,通过Plackett-Burman法对培养基组分筛选显著性影响因素,并对其培养基中无机盐进行正交试验优化,最后进行5 L发酵罐验证。【结果】最优培养基配方和培养条件:100 g/L葡萄糖,1.5 g/L尿素,0.20 g/L KH_2PO_4,0.20 g/L ZnSO_4,0.05 g/L MgSO_4,0.75 g/L KCl,30 g/L CaCO_3,0.01%吐温-80,发酵温度26°C,250 mL摇瓶装液量50 mL。【结论】通过优化,聚苹果酸的糖酸转化率达到0.71 g/g,生产强度达到0.89 g/(L·h),较优化前分别提高了18.33%和71.15%,为发酵葡萄糖合成聚苹果酸进而生产L-苹果酸工艺的工业化生产奠定经济性基础。     

Production of L(+)-lactic acid from glucose and starch by immobilized cells of Rhizopus oryzae in a rotating fibrous bed bioreactor

A rotating fibrous-bed bioreactor (RFB) was developed for fermentation to produce L(+)-lactic acid from glucose and cornstarch by Rhizopus oryzae. Fungal mycelia were immobilized on cotton cloth in the RFB for a prolonged period to study the fermentation kinetics and process stability. The pH and dissolved oxygen concentration (DO) were found to have significant effects on lactic acid productivity and yield, with pH 6 and 90% DO being the optimal conditions. A high lactic acid yield of 90% (w/w) and productivity of 2.5 g/L.h (467 g/h.m(2)) was obtained from glucose in fed-batch fermentation. When cornstarch was used as the substrate, the lactic acid yield was close to 100% (w/w) and the productivity was 1.65 g/L.h (300 g/h.m(2)). The highest concentration of lactic acid achieved in these fed-batch fermentations was 127 g/L. The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells. Without immobilization with the cotton cloth, mycelia grew everywhere in the fermentor and caused serious problems in reactor control and operation and consequently the fermentation was poor in lactic acid production. Oxygen transfer in the RFB was also studied and the volumetric oxygen transfer coefficients under various aeration and agitation conditions were determined and then used to estimate the oxygen transfer rate and uptake rate during the fermentation. The results showed that the oxygen uptake rate increased with increasing DO, indicating that oxygen transfer was limited by the diffusion inside the mycelial layer.     

Citric acid production by selected mutants of Aspergillus niger from cane molasses

The present investigation deals with citric acid production by some selected mutant strains of Aspergillus niger from cane molasses in 250 ml Erlenmeyer flasks. For this purpose, a conidial suspension of A. niger GCB-75, which produced 31.1 g/l citric acid from 15% (w/v) molasses sugar, was subjected to UV-induced mutagenesis. Among the 3 variants, GCM-45 was found to be a better producer of citric acid (50.0 +/- 2a) and it was further improved by chemical mutagenesis using N-methyl, N-nitro-N-nitroso-guanidine (MNNG). Out of 3,2-deoxy-D-glucose resistant variants, GCMC-7 was selected as the best mutant, which produced 96.1 +/- 1.5 g/l citric acid 168 h after fermentation of potassium ferrocyanide and H2SO4 pre-treated blackstrap molasses in Vogel's medium. On the basis of kinetic parameters such as volumetric substrate uptake rate (Qs), and specific substrate uptake rate (qs), the volumetric productivity, theoretical yield and specific product formation rate, it was observed that the mutants were faster growing organisms and produced more citric acid. The mutant GCMC-7 has greater commercial potential than the parental strain with regard to citrate synthase activity. The addition of 2.0 x 10(-5) M MgSO4 x 5H2O into the fermentation medium reduced the Fe2+ ion concentration by counter-acting its deleterious effect on mycelial growth. The magnesium ions also induced a loose-pelleted form of growth (0.6 mm, diameter), reduced the biomass concentration (12.5 g/l) and increased the volumetric productivity of citric acid monohydrate (113.6 +/- 5 g/l).     

Effects of neutralizing agents on lactic acid production by Rhizopus oryzae using sweet potato starch

A neutralizing agent is usually employed to counteract the pH reduction during lactic acid fermentation by Rhizopus oryzae. Calcium carbonate (CaCO₃) is used as such a pH controlling agent. The low solubility of CaCO₃ in the fermentation broth could however lead to low efficiency in pH control and cause problems in the subsequent purification process. Therefore, an alternative agent in place of CaCO₃ was examined in this study. The effect of four different neutralizing agents, including CaCO₃, sodium hydroxide (NaOH), ammoniacal solution and sodium bicarbonate (NaHCO₃) on lactic acid production and the morphology of the pellets were investigated. Results indicated that CaCO₃ was still the preferred choice, because of the pellet morphology and the highest lactic acid concentration (43.3 g/L) obtained in the batch using 60 g/L of sweet potato starch as feedstock. It is noteworthy that the lactic acid purification is relatively easier when using NaHCO₃ instead of CaCO₃, due to the higher solubility of sodium lactate than calcium lactate. Therefore, even the batch with CaCO₃ had a slightly higher productivity (1.23 g/L/h) than the batch with NaHCO₃ (1.14 g/L/h), NaHCO₃ might be the first choice for process designers whenever recovery is vital.     

Aerobic fungal cell immobilization in a dual hollow-fiber bioreactor: Continuous production of a citric acid

Aspergillus niger B60 was immobilized in a dual hollow-fiber bioreactor (DHFBR) to produce citric acid continuously. The fungi proliferated well in the interstitial region formed by a parallel arrangement of three microporous polypropylene hollow fibers contained within a silicone tube. Long-term operation with nitrogen-enriched medium was not possible due to expansion of the silicone tubes by continual cell growth. The fungal growth could be controlled by supplying a nitrogen-deficient medium at the production stage. With pure oxygen aeration and nitrogen-deficient medium, volumetric productivity reached 1.62 g/L h at a residence time of 4.02 h, which corresponded to a 27-fold increase over that of shake-flask fermentation. When the residence time was increased to 20.1 h, citric acid at a concentration of 26 g/L was continuously produced, with a yield of 80-90% and a volumetric productivity of 1.3 g/L h. This represents a significant improvement in final concentration, yield, and the volumetric productivity over the equivalent values of the corresponding batch fermentation, which were 18 g/L, 40%, and 0.06 g/L h, respectively.     

Fumaric acid production from xylose by immobilized Rhizopus arrhizus cells

Summary The production of fumaric acid by immobilized Rhizopus arrhizus TKK 204-1-1a mycelium was optimized in batch fermentations using statistical experimental design and empiric modelling. The maximum fumaric acid concentration was obtained at a xylose concentration of about 6% and a carbon:nitrogen ratio of about 160. In repeated batch fermentations with immobilized cells the highest volumetric productivity of fumaric acid reached was 87 mg/l per hour when the initial xylose concentration was 10%, the C:N ratio 160 and the residence time 1.75 days. The maximum product concentration was 16.4 g/l when the initial xylose concentration was 10%, the C:N ratio 160 and the residence time 10.25 days. The maximum yield from initial xylose (6.47%) was 23.7% with a product concentration of 15.3 g/l and volumetric productivity of 71 mg/l per hour at a residence time of 9 days and a C:N ratio of 188.3. Immobilization could increase the fumaric acid concentration to a level 3.4 times higher than that produced by free cells.     

基于Desirability函数法对米根霉发酵制备富马酸的多目标优化

以富马酸产量和生产速率为目标,通过正交实验考察了种龄,接种量,葡萄糖和尿素浓度对两者的影响,进一步利用基于响应曲面方法的Desirability函数确定了葡萄糖和尿素的最佳浓度。结果表明,最佳的种龄和接种量分别为36h和15%,葡萄糖和尿素的优化浓度为132.73和0.0586g/l,此时模型预测的富马酸产量和生产速率达到71.42g/l和0.804g/(l.h),Desirability的函数值高达0.966。该条件下在5L发酵罐水平上进行验证试验,经过88h的发酵最终生成富马酸66.5g/l,生产速率达到0.755 g/(l.h),与未优化前相比,产量和生产速率分别提高了13.9%和15.8%,取得了理想的效果,实现了产量和生产速率的同时优化,为发酵法制备富马酸的工业化放大奠定了基础。     

三相流化床L-乳酸发酵及与离子交换分离耦合

L—乳酸因其能被人体代谢而将在食品、医药工业中代替DL—乳酸。另外均聚和共聚L—乳酸在医学及生产可生物降解的包装材料、农膜等方面有巨大的潜在市场。L—乳酸一般采用米根霉     

Production of Fumaric Acid in 20-Liter Fermentors

The conditions necessary for the production of fumaric acid in 20-liter fermentors by fermentation of glucose with Rhizopus arrhizus strain NRRL 2582 were determined. Continuous neutralization of fumaric acid was necessary for optimal yields. Yields of the calcium salt were in excess of 65 g of fumaric acid from 100 g of sugar consumed during fermentation of sugar concentrations of 10 to 16%. Conditions established for calcium fumarate production include a simple mineral salts medium, 0.5 v:v:min aeration rate, 300 rev/min agitation rate in a baffled tank, 33 C incubation temperature, CaCO(3) to neutralize the acid formed, and a 4 to 5% (v/v) vegetative inoculum. A suitable procedure and medium for the preparation of a vigorous vegetative inoculum were established. The tendency for calcium fumarate fermentations to foam excessively was controlled with a proper antifoam agent added prior to sterilization of the medium and again at daily intervals during fermentation. The production of soluble sodium or potassium fumarates was inhibited when the concentration of fumarates reached 3.5 to 4.0%. No means of overcoming this inhibition was found. Starches and certain other grain-derived carbohydrates were fermented to form calcium fumarate in flask experiments with approximately the same efficiency as was glucose.     

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.     

Process optimization of continuous gluconic acid fermentation by isolated yeast-like strains of Aureobasidium pullulans

This study was focused on the optimization of a new fermentation process for continuous gluconic acid production by the isolated yeast-like strain Aureobasidium pullulans DSM 7085 (isolate 70). Operational fermentation parameters were optimized in chemostat cultures, using a defined glucose medium. Different optima were found for growth and gluconic acid production for each set of operation parameters. Highest productivity was recorded at pH values between 6.5 and 7.0 and temperatures between 29 and 31 degrees C. A gluconic acid concentration higher than 230 g/L was continuously produced at residence times of 12 h. A steady state extracellular gluconic acid concentration of 234 g/L was measured at pH 6.5. 122% air saturation yielded the highest volumetric productivity and product concentration. The biomass-specific productivity increased steadily upon raising air saturation. An intracellular gluconic acid concentration of about 159 g/L (0.83 mol) was determined at 31 degrees C. This is to be compared with an extracellular concentration of 223 g/L (1.16 mol), which indicates the possible existence of an active transport system for gluconic acid secretion, or the presence of extracellular glucose oxidizing enzymes. The new process provides significant advantages over the traditional discontinuous fungi operations. The process control becomes easier, thus offering stable product quality and quantity.