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.     

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.     

Enhanced production of L-(+)-lactic acid in chemostat by Lactobacillus casei DSM 20011 using ion-exchange resins and cross-flow filtration in a fully automated pilot plant controlled via NIR

Due to the lack of suitable in-process sensors, on-line monitoring of fermentation processes is restricted almost exclusively to the measurement of physical parameters only indirectly related to key process variables, i.e., substrate, product, and biomass concentration. This obstacle can be overcome by near infrared (NIR) spectroscopy, which allows not only real-time process monitoring, but also automated process control, provided that NIR-generated information is fed to a suitable computerized bioreactor control system. Once the relevant calibrations have been obtained, substrate, biomass and product concentration can be evaluated on-line and used by the bioreactor control system to manage the fermentation. In this work, an NIR-based control system allowed the full automation of a small-scale pilot plant for lactic acid production and provided an excellent tool for process optimization. The growth-inhibiting effect of lactic acid present in the culture broth is enhanced when the growth-limiting substrate, glucose, is also present at relatively high concentrations. Both combined factors can result in a severe reduction of the performance of the lactate production process. A dedicated software enabling on-line NIR data acquisition and reduction, and automated process management through feed addition, culture removal and/or product recovery by microfiltration was developed in order to allow the implementation of continuous fermentation processes with recycling of culture medium and cell recycling. Both operation modes were tested at different dilution rates and the respective cultivation parameters observed were compared with those obtained in a conventional continuous fermentation. Steady states were obtained in both modes with high performance on lactate production. The highest lactate volumetric productivity, 138 g L(-1) h(-1), was obtained in continuous fermentation with cell recycling.     

A near-infrarod spectroscopy technique for the control of fermentation processes: An application to lactic acid fermentation

A near-infrared (NIR) spectroscopy technique for the control of lactic acid fermentation process has been proposed. Lactic acid, glucose, and biomass concentrations were determined by the NIR spectroscopy method. The three parameters examined were closely correlated to the results obtained with classical laboratory procedures. Moreover, the conditions for the on-line utilization of the NIR spectroscopy measurement system were pointed out. The great versatility of the NIR spectroscopy should permit its use for other fermentation processes. (c) 1994 John Wiley & Sons, Inc.     

响应面法优化北五味子褐斑病内生生防真菌淡紫拟青霉WG9发酵工艺及发酵产物稳定性研究

为提高北五味子内生生防真菌WG9发酵液对褐斑病病原菌的抑菌率,本研究采用单因素实验及响应面法对其发酵条件进行优化.首先采用单因素实验,以发酵时间(h)、发酵温度(℃)、摇床转速(r/min)及初始pH为考察指标,测定对病原菌的抑菌率及生防真菌WG9的菌丝体生物量,以获取单因素实验的最优值,然后使用Box-Behnken...     

拉曼光谱分析技术在资源微生物发酵性能评价中的应用

微生物发酵过程是细胞新陈代谢进行物质转化的过程,为了提高目标产物的转化率,需要对微生物发酵动态特性进行实时分析,以便实时优化发酵过程。拉曼光谱(Raman spectroscopy)量化测试作为一种有应用前景的在线过程分析技术,可以在避免微生物污染的条件下,实现精准监测,进而用于优化控制微生物发酵过程。【目的】以运动发酵单胞菌(Zymomonas mobilis)为例,建立微生物发酵过程中葡萄糖、木糖、乙醇和乳酸浓度拉曼光谱预测模型,并进行准确性验证。【方法】采用浸入式在线拉曼探头,收集运动发酵单胞菌发酵过程中多个组分的拉曼光谱,采用偏最小二乘法对光谱信号进行预处理和多元数据分析,结合离线色谱分析数据,对拉曼光谱进行建模分析和浓度预测。【结果】针对运动发酵单胞菌,首先实现拉曼分析仪对单一产品乙醇发酵过程的精准检测,其次基于多元变量分析,建立葡萄糖、乙醇和乳酸浓度变化的预测模型,实现对发酵过程中各成分浓度变化的准确有效分析。【结论】成功建立了一种评价资源微生物尤其是工业菌株发酵液多种组分的拉曼光谱分析方法。该方法为运动发酵单胞菌等工业菌株利用多组分底物工业化生产不同产物的实时检测,以及其他微生物尤其工业菌株的选育和过程优化提供了新方法。     

Effects of acid pre-treatment on bio-hydrogen production and microbial communities during dark fermentation

Optimal conditions for acid pre-treatment were investigated for the enrichment of hydrogen-producing bacteria (HPB) in a mixed culture using three strong acids: HCl, HNO(3), and H2SO4 x HCl was selected as a suitable acid for the enrichment of HPB in the fermentation process. The volume of bio-hydrogen produced when the mixed culture was pre-treated using HCl at pH 2 was 3.2 times higher than that obtained without acid pre-treatment. Changes in the microbial community during acid pre-treatment were monitored using images obtained by the fluorescent in situ hybridization (FISH) method and the Live/Dead cell viability test. The tests clearly indicated that the Clostridium species of cluster I were the predominant strains involved in bio-H(2) fermentation, and could be selectively enriched by HCl pre-treatment.     

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     

Bioconversion of grape must into modulated gluconic acid production by Aspergillus niger ORS-4.410

AIMS: Analysis of regulators for modulated gluconic acid production under surface fermentation (SF) condition using grape must as the cheap carbohydrate source, by mutant Aspergillus niger ORS-4.410. Replacement of conventional fermentation condition by solid-state surface fermentation (SSF) for semi-continuous production of gluconic acid by pseudo-immobilization of A. niger ORS-4.410. METHODS AND RESULTS: Grape must after rectification was utilized for gluconic acid production in batch fermentation in SF and SSF processes using mutant strain of A. niger ORS-4.410. Use of rectified grape must led to the improved levels of gluconic acid production (80-85 g l(-1)) in the fermentation medium containing 0.075% (NH4)2HPO4; 0.1% KH2PO4 and 0.015% MgSO4.7H2O at an initial pH 6.6 (+/-0.1) under surface fermentation. Gluconic acid production was modulated by incorporating the 2% soybean oil, 2% starch and 1% H2O2 in fermentation medium at continuously high aeration rate (2.0 l min(-1)). Interestingly, 95.8% yield of gluconic acid was obtained when A. niger ORS-4.410 was pseudo-immobilized on cellulose fibres (bagasse) under SSF. Four consecutive fermentation cycles were achieved with a conversion rate of 0.752-0.804 g g(-1) of substrate into gluconic acid under SSF. CONCLUSIONS: Use of additives modulated the gluconic acid production under SF condition. Semi-continuous production of gluconic acid was achieved with pseudo-immobilized mycelia of A. niger ORS-4.410 having a promising yield (95.8%) under SSF condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioconversion of grape must into modulated gluconic acid production under SSF conditions can further be employed in fermentation industries by replacing the conventional carbohydrate sources and expensive, energy consuming fermentation processes.     

A Computer-aided noninterfering on-line technique for monitoring oxygen-transfer characteristics during fermentation processes

The role of computers in the monitoring and control of fermentation processes has increased steadfastly. The ultimate utility of the machines will not depend on the availability of online sensors but also on the availability of techniques that combine direct measurements, leading towards estimates of variable closely related to the microbial process or its control. In this article, a methodology for on-line and noninterfering evaluation of the volumetric mass-transfer coefficient K(l)a is developed. A detailed presentation of the procedure, called "the static method," is given. Its feasibility is proved through implementation of the method on an antibiotic fermentation process. These experiments indicate that operator actions meant to modify the oxygen-transfer conditions can be checked on-line. The quantitative value of the static method is ascertained by comparing the experimental results with K(l)a estimates obtained with the "gassing-out" method. A sensitivity analysis was carried out, revealing the need for temperature and pressure corrections and showing that the precision of the oxygen analyzer determines the precision of the static method.     

Assessment of the yeast species composition of cocoa bean fermentations in different cocoa-producing regions using denaturing gradient gel electrophoresis

The yeast species composition of 12 cocoa bean fermentations carried out in Brazil, Ecuador, Ivory Coast and Malaysia was investigated culture-independently. Denaturing gradient gel electrophoresis of 26S rRNA gene fragments, obtained through polymerase chain reaction with universal eukaryotic primers, was carried out with two different commercial apparatus (the DCode and CBS systems). In general, this molecular method allowed a rapid monitoring of the yeast species prevailing during fermentation. Under similar and optimal denaturing gradient gel electrophoresis conditions, the CBS system allowed a better separated band pattern than the DCode system and an unambiguous detection of the prevailing species present in the fermentation samples. The most frequent yeast species were Hanseniaspora sp., followed by Pichia kudriavzevii and Saccharomyces cerevisiae, independent of the origin of the cocoa. This indicates a restricted yeast species composition of the cocoa bean fermentation process. Exceptionally, the Ivorian cocoa bean box fermentation samples showed a wider yeast species composition, with Hyphopichia burtonii and Meyerozyma caribbica among the main representatives. Yeasts were not detected in the samples when the temperature inside the fermenting cocoa pulp-bean mass reached values higher than 45 °C or under early acetic acid production conditions.     

Image analysis for monitoring the barley tempeh fermentation process

AIMS: To develop a fast, accurate, objective and nondestructive method for monitoring barley tempeh fermentation. METHODS AND RESULTS: Barley tempeh is a food made from pearled barley grains fermented with Rhizopus oligosporus. Rhizopus oligosporus growth is important for tempeh quality, but quantifying its growth is difficult and laborious. A system was developed for analysing digital images of fermentation stages using two image processing methods. The first employed statistical measures sensitive to image colour and surface structure, and these statistical measures were highly correlated (r=0.92, n=75, P<0.001) with ergosterol content of tempeh fermented with R. oligosporus and lactic acid bacteria (LAB). In the second method, an image-processing algorithm optimized to changes in images of final tempeh products was developed to measure number of visible barley grains. A threshold of 5 visible grains per Petri dish indicated complete tempeh fermentation. When images of tempeh cakes fermented with different inoculation levels of R. oligosporus were analysed the results from the two image processing methods were in good agreement. CONCLUSION: Image processing proved suitable for monitoring barley tempeh fermentation. The method avoids sampling, is nonintrusive, and only requires a digital camera with good resolution and image analysis software. SIGNIFICANCE AND IMPACT OF THE STUDY: The system provides a rapid visualization of tempeh product maturation and qualities during fermentation. Automated online monitoring of tempeh fermentation by coupling automated image acquisition with image processing software could be further developed for process control.     

Application of dynamic calorimetry for monitoring fermentation processes

The rate of heat evolution (kcal/liter-hr) in mycelial fermentations for novobiocin and cellulase production with media containing noncellular solids was measured by an in situ dynamic calorimetric procedure. Thermal data so obtained have proved significant both in monitoring cell concentration during the trophophase (growth phase) and in serving as a physiological variable in the fermentation process. The validity of this technique has been demonstrated by closing the overall material and energy balances. The maintenance energy in a batch fermentation can also be calculated by integrating heat evolution data. This integration method is applicable to a fermentation lacking a precise cell growth curve. The maintenance coefficient, obtained for the novobiocin fermentation by Streptomyces niveus, is equal to 0.028 g glucose equivalent/g cell-hr. The production of novobiocin in the idio-phase (production phase) also correlates well with the amount of energy catabolixed for maintenance and this results in an observed conversion yield of glucose to novobiocin of 11.8 mg of novobiocin produced per gram of glucose catabolized. A new physiological variable, kilocalories of heat evolved per millimole of oxygen consumed, has been proposed to monitor the state of cells during the fermentation. This method may provide a simple way to monitor on-line shifts in the efficiency of cell respiration and changes in growth yields during a microbial process.     

油酸促进灵芝三萜液态深层发酵的工艺研究及规模化验证

灵芝三萜是灵芝中主要的活性成分之一,前期研究发现油酸可以促进灵芝三萜液态深层发酵下的发酵合成。本研究主要对油酸促进灵芝三萜液态深层发酵的工艺进行优化,并进行3L发酵罐规模的验证。通过单因素实验考察油酸的添加方式、添加时间和添加浓度对灵芝三萜的影响,结合响应面实验,获得最优工艺条件并进行验证：在发酵第32h添加1.21%高温灭菌油酸,最高灵芝三萜含量为42.69mg/g;在发酵第7h添加1.35%过滤除菌油酸,最高三萜含量为43.38mg/g,分别比对照提高2.04倍和2.08倍。在1 000mL摇瓶中添加高温灭菌油酸和过滤除菌油酸,灵芝三萜含量分别为32.18和32.48mg/g,为对照的1.96倍和1.95倍;在3L发酵罐规模下灵芝三萜含量分别为28.66和25.13mg/g,为对照的1.62倍和1.42倍。本研究系统优化了油酸促进灵芝三萜液态深层发酵的工艺条件,并在与工业生产相对应的3L发酵罐上进行验证。该研究可为灵芝三萜的规模化发酵提供重要参考和借鉴。     

茴香酸产生菌发酵条件的优化

为提高微生物降解反式茴脑获得茴香酸的产量,对假单胞菌Pseudomonas sp.NT2的发酵参数进行优化,以提高降解过程的转化率。利用单因素试验考察碳氮源种类及浓度、反式茴脑添加量、发酵温度、接种量、初始pH以及装液量对茴香酸生成量、反式茴脑降解率的影响,通过Plackett-Burman试验和最陡爬坡试验确定影响茴香酸生成量的显著因素并获取中心点,最后采用Box-Behnken模型进行响应面优化得到最佳发酵条件并验证。结果表明氯化铵浓度、初始pH和装液量是显著影响因素,最佳发酵条件为:柠檬酸钠10 g/L,氯化铵1.26 g/L,反式茴脑添加量1%,发酵温度30℃,接种量4%,初始pH 7.9,装液量42 mL/250 mL。优化后茴香酸生成量为7.24 g/L,为优化前的3.5倍,茴香酸摩尔生成率为80.72%,反式茴脑降解率为89.81%,分别比优化前提高了270.28%和97.78%。综上,假单胞菌NT2是生物转化生产茴香酸的潜力菌株。响应面优化可以显著提高反式茴脑的降解率和茴香酸产量,这为大规模生产茴香酸奠定了基础。     

Analysis of metabolic fluxes for hyaluronic acid (HA) production by Streptococcus zooepidemicus

Summary A detailed metabolic flux analysis (MFA) for hyaluronic acid (HA) production by Streptococcus zooepidemicus was carried out. A metabolic network was constructed for the metabolism of S. zooepidemicus. Fluxes through these reactions were estimated by MFA using accumulation rates of biomass and product, consumption rate of glucose in batch fermentation and dissolved oxygen-controlled fermentation. The changes of the fluxes were observed at different stages of batch fermentation and in different dissolved oxygen tension (DOT)-controlled fermentation processes. The effects of metabolic nodes on HA accumulation under various culture conditions were investigated. The results showed that high concentration of glucose in the medium did not affect metabolic flux distribution, but did influence the uptake rate of glucose. HA synthesis was influenced by DOT via flux redistribution in the principal node. Adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) produced in the fermentation process are associated with cell growth and HA synthesis.     

State analysis of fermentation using a mass spectrometer with membrane probe

A new method of continuous process analysis in fermentation using a mass spectrometer (MS) membrane probe is described. A number of samples from industrial fermentations were analyzed for the occurrence of volatiles detectable with a silicone membrane probe connected to a quadrupole MS. In all fermentations, characteristic spectra were observed which were found to change systematically during the batch process. Factor analysis was used to treat the data. The factor scores were compared with the actual product concentrations (antibiotics, toxins, etc.), which were measured using other analytical methods and were found to correlate with them. On-line analysis was also carried out on a fermentation with an MS and an Apple II microcomputer. Direct monitoring of products, which are not directly measurable with the membrane MS probe requires a new calibration each time conditions such as medium composition are changed.     

Microbial pretreatment of coals: A tool for solubilization of lignite in organic solvent – quinoline

A method was developed for the conversion of low rank coals to products soluble in an organic solvent (quinoline). A selected group of polynuclear aromatic-compound-degrading and lignin-degrading facultative pure cultures and enriched anaerobic mixed microbial cultures developed for this purpose were used separately as well as together under co-culture conditions for stepwise treatment of Neyveli lignite (NL). This aerobic-anaerobic co-metabolic (co-culture) biodegradation (AACB) process resulted in the enhancement of quinoline extractability of the lignite, thereby yielding clean coal substance (the extract). The residual lignite obtained after quinoline extraction was subjected to a second step of AACB fermentation treatment. This resulted in further extraction of lignite in quinoline. The conditions were optimised for AACB fermentation treatment. The two-step AACB fermentation process under optimum conditions, resulted in an overall enhancement of yield of extract from 18% for the original lignite sample to 56% for the treated sample. The changes in the filtrate were evaluated using UV spectra, those in the residue were evaluated using FTIR spectroscopy and UV-reflectance and those in the extract using proton NMR spectra of the chloroform soluble fraction. The results indicated a decreased absorption in the carbonyl region in the AACB-treated residue and also a decrease in the overall mineral matter in the lignite samples. The mechanism of the AACB fermentation process is discussed. The process affords biosolubilization of lignite in organic solvent (quinoline) under milder conditions along with a simultaneous removal of a part of the mineral matter present in the coal. Uses for the clean coal extract obtained are suggested.     

Integrated process of starch ethanol and cellulosic lactic acid for ethanol and lactic acid production

The sequential production of bioethanol and lactic acid from starch materials and lignocellulosic materials was investigated as ethanol fermentation broth (EFB) can provide nutrients for lactic acid bacteria. A complete process was developed, and all major operations are discussed, including ethanol fermentation, broth treatment, lactic acid fermentation, and product separation. The effect of process parameters, including ethanol fermentation conditions, treatment methods, and the amount of EFB used in simultaneous saccharification and fermentation (SSF), is investigated. Under the selected process conditions, the integrated process without additional chemical consumption provides a 1.08 acid/alcohol ratio (the broth containing 22.4 g/L ethanol and 47.6 g/L lactic acid), which corresponds to a polysaccharide utilization ratio of 86.9 %. Starch ethanol can thus promote cellulosic lactic acid by providing important nutrients for lactic acid bacteria, and in turn, cellulosic lactic acid could promote starch ethanol by improving the profit of the ethanol production process. Two process alternatives for the integration of starch ethanol and cellulosic lactic acid are compared, and some suggestions are given regarding the reuse of yeast following the cellulosic SSF step for lactic acid production.     

Evidence for nitrogen catabolite repression during citric acid production by Aspergillus niger under phosphate-limited growth conditions

Experiments performed in batch fermentation under phosphate-limited growth conditions showed that the citric acid yield was inversely related to the excess nitrogen concentration in the medium. Results from chemostat culture confirmed a negative relationship between the citric acid yield and both the specific growth rate and the nitrogen consumption rate. This is evidence for nitrogen catabolite repression. A fed-batch fermentation performed under dual phosphate/nitrogen limitation produced results very similar to those from a culture limited by nitrogen alone. There is no advantage in maintaining an excess of phosphate during citric acid production and the process will therefore be more economic when operated under dual limitation conditions.