A new approach of pellet formation of a filamentous fungus -Rhizopus oryzae

The effects of inoculum and medium composition (i.e. potato dextrose broth as carbon source, soybean peptone, calcium carbonate, and metal ions) on pellet formation of Rhizopus oryzae ATCC 20344 have been studied. Metal ions were found to have a significant negative effect on pellet formation while soybean peptone had a positive effect. In addition, potato dextrose broth and calcium carbonate were beneficial to R. oryzae for growing small, smooth pellets during the culture. The study also demonstrated that an inoculum size of less than 1.5x10(9)spores/L had no significant influence on pellet formation although it had large impacts on pellet growth. Thus, a new approach to form pellets has been developed using only potato dextrose broth, soybean peptone, and calcium carbonate allowing for pellet size to be controlled by adjusting inoculum size and the concentrations of potato dextrose broth, soybean peptone, and calcium carbonate in the medium.     

Copper accumulation and phosphatase activities of Aspergillus and Rhizopus

Copper accumulation and phosphatase activities of three Aspergillus species resistant to copper were compared to three copper-sensitive Rhizopus species. High level of acid phosphatases and decreased Cu2+-uptake were found with resistant in contrast to sensitive strains. The presence of copper(II) ions in the medium increased the production of acid phosphatases in the resistant A. niger and decreased their activity in the sensitive R. delemar. Copper ions inhibited the activity of A. niger cellular acid phosphatase with a Ki of 8.9x10(-4) M and slightly activated the R. delemar enzyme.     

Growth ofRhizopus arrhizus in fermentation media

Summary Rhizopus arrhizus biomass attached itself to fermentor walls, baffles and impellers when grown in casein/ glucose media. In shake flasks, dispersed filamentous growth was produced in media containing certain concentrations of glucose and soya flour. Other media tested produced pelleted or clumpy growth. Medium initial pH did not affect morphology type. Dispersed growth could not be obtained by addition of detergents, oils and polymers to a clear glucose/soya peptone medium. Addition of maize solids to this medium resulted in dispersed growth which occurred even in the presence of calcium, which in most media caused pellet formation. Mycelia appeared to bind to the maize particles and use these as growth centres thereby preventing pellet or clump formation. Mycelial pellets appeared to originate either from a single spore or by interaction of branched hyphae from different spores. Medium composition and macro-morphology type correlate with differences in hyphal structures.     

Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae

Dicarboxylic acids that are produced from renewable resources are becoming attractive building blocks for the polymers industry. In this respect, fumaric acid is very interesting. Its low aqueous solubility facilitates product recovery. To avoid excessive waste salt production during downstream processing, a low pH for fumaric acid fermentation will be beneficial. Studying the influence of pH, working volume and shaking frequency on cell cultivation helped us to identify the best conditions to obtain appropriate pellet morphologies of a wild type strain of Rhizopus oryzae. Using these pellets, the effects of pH and CO(2) addition were studied to determine the best conditions to produce fumaric acid in batch fermentations under nitrogen-limited conditions with glucose as carbon source. Decreasing either the fermentation pH below 5 or increasing the CO(2) content of the inlet air above 10% was unfavourable for the cell-specific productivity, fumaric acid yield, and fumaric acid titer. However, switching off the pH control late in the batch phase did not affect these performance parameters and allowed achieving pH of 3.6. A concentration of 20 gL(-1) of fumaric acid was obtained at pH 3.6 while the average cell mass specific productivity and fumaric acid yield were the same as at pH 5.0. Consequently, relatively modest amounts of inorganic base were required for pH control, while recovery of the acid should be relatively easy at pH 3.6.     

Removal of heavy metals from aqueous solution using Rhizopus delemar mycelia in free and polyurethane-bound form

This study assesses the ability of mycelia of Rhizopus delemar (both free and immobilized on polyurethane foam) to remove heavy metals from single-ion solutions as well as from a mixture of them. All experiments were conducted using 0.5-5 mm solutions of CuSO4 x 5H2O, CoCl2-6H2O and FeSO4 7H2O. Mycelia immobilized on polyurethane foam cells showed some times increase in uptake compared with that of free cells. Metal ions accumulation from a mixed solution was decreased slightly for cobalt and iron and considerable for copper ions. Heavy metal uptake was examined in the immobilized column experiments and more than 92% heavy metal removal (mg heavy metals removed/mg heavy metals added) from a mixed solution was achieved during the 5 cycles. During these experiments, the dry weight of the immobilized cells was decreased by only 2%. These results showed that immobilized mycelia of Rhizopus delemar can be used repeatedly for removal of heavy metals from aqueous solutions.     

Production of geranyl acetate and other acetates by direct esterification catalyzed by mycelium of Rhizopus delemar in organic solvent

Dry mycelium of Rhizopus delemar MIM catalyzed the formation of geranyl acetate using 110 mM geraniol and acetic acid at 55°C in heptane to give 11.9 g/l (55% molar conversion). Geranyl acetate was produced at 72.5-75 g/l after 10 days by semi-continuous addition of the substrates. Rhizopus delemar also catalyzed the direct acetylation of different primary alcohols with molar conversions ranging from 65 to 98%.     

Production of lactic acid and byproducts from waste potato starch by <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis>: role of nitrogen sources

Summary Lactic acid was produced by Rhizopus arrhizus using waste potato starch as the substrate. The aim of this study was to identify the role of nitrogen sources and their impact on the formation of lactic acid and associated byproducts. Ammonium sulphate, ammonium nitrate, urea, yeast extract and peptone were assessed in conjunction with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio favoured the production of fumaric acid. Ammonium nitrate appeared to be the most suitable nitrogen source for achieving a high and stable lactic acid yield, and minimizing the production of byproducts such as biomass and ethanol, while urea proved to be the least favourable nitrogen source. Yeast extract and peptone appeared to improve fungal cell growth. The kinetics data revealed that a high concentration of ammonium nitrate enhanced the lactic acid productivity. The maximum lactic acid concentration of 36.4 g/l, representing a yield of 91%, was obtained with addition of 0.909 g/l ammonium nitrate in 32 h.     

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.     

Optimization of fumaric acid production from potato flour by Rhizopus arrhizus

Summary Fumaric acid production by Rhizopus arrhizus from potato flour was studied at different initial substrate concentrations (S), C/N ratios and fermentation times (t f) in a composite design experiment. By using response surface methodology and canonical analysis, the experimental values of fumaric acid and mycelial biomass yields and productivity were fitted to the only statistically significant factors with mean percentage errors of 11, 26 and 13%, respectively. Owing to the limited statistical significance of the C/N ratio, it was possible to determine the values of S (25–50 g/l of glucose equivalent) and t f(44–100 h) associated with fumaric acid yields ranging from 60 to 75% and productivities varying from 6 to 8.4 g/l per day. Since such results were in quite good agreement with previous experiments carried out on other starchy materials, the above operating conditions might be used to minimize fumaric acid production costs as a function of the feed-stock used. Offsprint requests to: M. Moresi     

Metabolic engineering of Rhizopus oryzae: Effects of overexpressing pyc and pepc genes on fumaric acid biosynthesis from glucose

Fumaric acid, a dicarboxylic acid used as a food acidulant and in manufacturing synthetic resins, can be produced from glucose in fermentation by Rhizopus oryzae. However, the fumaric acid yield is limited by the co-production of ethanol and other byproducts. To increase fumaric acid production, overexpressing endogenous pyruvate carboxylase (PYC) and exogenous phosphoenolpyruvate carboxylase (PEPC) to increase the carbon flux toward oxaloacetate were investigated. Compared to the wild type, the PYC activity in the pyc transformants increased 56%-83%, whereas pepc transformants exhibited significant PEPC activity (3-6mU/mg) that was absent in the wild type. Fumaric acid production by the pepc transformant increased 26% (0.78g/g glucose vs. 0.62g/g for the wild type). However, the pyc transformants grew poorly and had low fumaric acid yields (<0.05g/g glucose) due to the formation of large cell pellets that limited oxygen supply and resulted in the accumulation of ethanol with a high yield of 0.13-0.36g/g glucose. This study is the first attempt to use metabolic engineering to modify the fumaric acid biosynthesis pathway to increase fumaric acid production in R. oryzae.     

l-Malic Acid Fermentation by Mixed Culture of Rhizopus arrhizus and Proteus vulgaris

When Rhizopus arrhizus NRRL 1526 was mix-cultured with Proteus vulgaris AHU 1144, a strain having a high fumarase activity, in a medium containing glucose as a substrate, fumaric acid fermentation was successively converted to l-malic acid fermentation and large amounts of l-malic acid were accumulated as an end product.

As an inoculum of P. vulgaris for this fermentation, cells in the stationary growth phase (48 to 72 hr culture) were much more favorable than those in the exponential growth phase (18 hr culture) and malic acid yields in the former case were as high as about 70 to 75 % based on initial glucose after 3 to 4 days of the mixed culture.     

An optimized fed-batch culture strategy integrated with a one-step fermentation improves <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid production by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

In previous work, we proposed a novel modified one-step fermentation fed-batch strategy to efficiently generate l-lactic acid (l-LA) using Rhizopus oryzae. In this study, to further enhance efficiency of l-LA production through one-step fermentation in fed-batch cultures, we systematically investigated the initial peptone- and glucose-feeding approaches, including different initial peptone and glucose concentrations and maintained residual glucose levels. Based on the results of this study, culturing R. oryzae with initial peptone and glucose concentrations of 3.0 and 50.0 g/l, respectively, using a fed-batch strategy is an effective approach of producing l-LA through one-step fermentation. Changing the residual glucose had no obvious effect on the generation of l-LA. We determined the maximum LA production and productivity to be 162 g/l and 6.23 g/(l·h), respectively, during the acid production stage. Compared to our previous work, there was almost no change in l-LA production or yield; however, the productivity of l-LA increased by 14.3%.     

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.     

戴尔根霉发酵麸皮酶解液制备富马酸的研究

拟通过根霉菌发酵小麦麸皮纤维质实现其高效生物转化制备富马酸的目标。在单因素试验研究基础上,对发酵工艺进行响应曲面法优化,并开展代谢机理初步探索。通过单因素试验确定了酵母浸粉、硫酸镁及硫酸锰质量浓度为主要影响因素,响应曲面研究结果显示:当发酵水解液总糖浓度80.0g/L、硫酸铵2.00g/L、酵母浸粉0.29g/L、硫酸镁0.26g/L、硫酸锰0.07g/L、硫酸亚铁0.05g/L时,富马酸产量最高,其值为27.423g/L。对戴尔根霉Rhizopus delemar CICC41341的木糖代谢途径的初步分析结果表明木酮糖激酶为该菌株木糖代谢的关键限速酶。论文研究结果可为纤维质糖液工业发酵制备平台有机酸提供一定支持。     

Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique

Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).     

米根霉ME—F12发酵产富马酸的菌体形态控制

丝状真菌发酵体系中菌体形态对产量有着重要影响。考察富马酸产生菌Rhizopus oryzae ME—F12种子培养过程中不同pH条件、孢子悬浮液密度以及CaCl2添加量对其形态的影响。结果表明,当控制种子培养液pH2．3～2．7、接种孢子的终密度为1．5×10^8～3．0×10^8／L和添加0．5g／LCaCl2时,培养可获得直径约为0．65mm光滑规整茵球,后继的产酸发酵中富马酸量高达58．9g／L。正交实验表明,pH是影响菌球形成的最主要因素,孢子液密度主要影响菌体生物量,而CaCl2则是菌球表面光滑度的主要影响因素。     

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。     

Synthesis of various kinds of esters by four microbial lipases

Ester synthesis by microbial lipases, using homogeneous enzyme preparations, were investigated. The amount of synthesized ester was estimated by alkalimetry, and products were identified by thin-layer chromatography and infrared spectroscopy. Lipases from Aspergillus niger, Rhizopus delemar, Geotrichum candidum and Penicillium cyclopium synthesized esters from oleic acid and various primary alcohols. Only Geotrichum candidum lipase synthesized esters of secondary alcohols. Esters of tertiary alcohols, phenols or sugar alcohols were not synthesized by any lipase. Rather high concentrations of alcohol were required to synthesize the esters of ethylene glycol, propylene glycol or trimethylene glycol. Lipases from Aspergillus niger and Rhizopus delemar synthesized oleyl esters of various fatty acids and some dibasic acids. In contrast, lipases from Geotrichum candidum and Penicillium cyclopium synthesized oleyl esters only from medium or long chain fatty acids.     

积累L-苹果酸的米根霉突变株酶活性初探

对富马酸产生菌株—米根霉ME-F10进行诱变育种的过程中, 得到一株性能稳定的高效积累L-苹果酸的突变株ME-M15。该菌株发酵96 h平均L-苹果酸产量达16.3 g/L, 较出发菌株L-苹果酸积累量平均提高3倍, 而富马酸和乙醇的积累量大幅下降。对突变株代谢途径关键酶活研究表明, 突变株富马酸酶胞质途径同功酶和乙醇脱氢酶活力较之出发菌株酶活力明显减弱, 而丙酮酸羧化酶活力无明显差别。     

Fumaric Acid Production by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> ATCC® 20344™ from Lignocellulosic Syrup

Powdered activated carbon-treated lignocellulosic syrup prepared from energy cane bagasse was evaluated as a potential feedstock in the production of fumaric acid by Rhizopus oryzae ATCC® 20344?. Energy cane bagasse was pretreated with dilute ammonia and enzymatically hydrolyzed with commercially available enzymes, Cellic® CTec2 and HTec2. The collected hydrolysate samples were subjected to powdered activated carbon adsorption for the removal of non-sugar compounds (i.e., organic acids, furaldehydes, total phenolic compounds) and concentrated to a final 65°Bx syrup (mostly xylose and glucose sugars). The use of lignocellulosic syrup, the effect of nitrogen source, medium additives, and initial pH in the seed culture medium on fungal morphology were investigated. The carbon to nitrogen (C/N) ratio in the acid production medium was also optimized for maximum yields in fumaric acid production. Optimum seed culture medium conditions (2.0 g/L urea, 3.0 pH) produced the desired compact, smooth, and uniform fungal pellets. Optimum acid production medium conditions (400 C/N ratio, 0.2 g/L urea) resulted in a fumaric acid production of 34.20 g/L, with a yield of 0.43 g/g and a productivity of 0.24 g/L/h. These results were comparable to those observed with the control medium (pure glucose and xylose). The present study demonstrated that lignocellulosic syrup processed from dilute ammonia pretreated energy cane bagasse has potential as a renewable carbon source for fumaric acid fermentation by Rhizopus oryzae ATCC® 20344?.