High-yield production of oxalic acid for metal leaching processes by Aspergillus niger

Abstract The complex-forming compound oxalic acid can effectively solubilise metals such as aluminium, iron, lithium, and manganese. In order to produce high amounts of oxalic acid for biohydrometallurgical processes, it was the aim of this work to optimise oxalic acid production by Aspergillus niger , a fungus well known for its ability to produce oxalic acid. A. niger excreted 427 mmol oxalic acid 1⁻¹ if it was cultivated in a pH-controlled (pH 6.0) fed-batch run in a 2-1 stirred tank reactor. Sucrose and lactose permeate were suitable carbon sources for oxalic acid production. In sucrose medium, A. niger produced high amounts of gluconic and oxalic acids, whereas in lactose permeate medium only oxalic acid was produced. Cultivation in green syrup and molasses media lead to high yields of biomass, but low oxalic acid production (<20 mmol 1⁻¹).     

Construction and characterization of an oxalic acid nonproducing strain of Aspergillus niger

Aspergillus niger produces oxalic acid as a by-product which causes problems with downstream processing of industrial enzymes. To overcome this problem the oah gene encoding oxaloacetate hydrolase (EC 3.7.1.1) was disrupted in a glucoamylase-producing strain of A. niger and the resulting strain was incapable of producing oxalic acid. The strain with the disrupted gene was compared with the wild-type strain producing oxalic acid in batch cultivations. The specific growth rate of both strains was 0.20 h(-1). The citric acid yields were identical, but the glucoamylase yield was only 50% in the disruptant compared with the wild-type strain. Batch experiments with 13C-labeled glucose as substrate were carried out to determine the metabolic fluxes through the central metabolism. The two strains had almost identical metabolic fluxes, which suggested that it was possible to disrupt the oah gene without pleiotropic consequences. The flux through the pentose phosphate pathway was around 60% of the glucose uptake for both strains, which suggested that a sufficient supply of NADPH was available for biosynthesis.     

Comparing phosphorus mobilization strategies using Aspergillus niger for the mineral dissolution of three phosphate rocks

Phosphorus deficiencies are limiting crop production in agricultural soils worldwide. Locally available sources of raw phosphate rock (PR) are being recognized for their potential role in soil fertility improvement. Phosphorus bioavailability is essential for the efficiency of PRs and can be increased by acid treatments. The utilization of organic acid producing micro-organisms, notably Aspergillus niger , presents a sustainable alternative to the use of strong inorganic acids, but acid production of A. niger strongly depends on the mineral content of the growth media. This study compared the phosphorus mobilization efficiency of two biological treatments, namely addition of acidic cell-free supernatants from A. niger cultivations to PRs and the direct cultivation of A. niger with PRs. The results show that addition of PR to cultivations leads to significant differences in the profile of organic acids produced by A. niger . Additions of PR, especially igneous rocks containing high amounts of iron and manganese, lead to reduced citric acid concentrations. In spite of these differences, phosphorus mobilization was similar between treatments, suggesting that the simpler direct cultivation method was not inferior. In addition to citric acid, it is suggested that oxalic acid contributes to PR solubilization in direct cultivations with A. niger , which would benefit farmers in developing countries where conventional fertilizers are not adequately accessible.     

Identification of fungal oxaloacetate hydrolyase within the isocitrate lyase/PEP mutase enzyme superfamily using a sequence marker-based method

Aspergillus niger produces oxalic acid through the hydrolysis of oxaloacetate, catalyzed by the cytoplasmic enzyme oxaloacetate acetylhydrolase (OAH). The A. niger genome encodes four additional open reading frames with strong sequence similarity to OAH yet only the oahA gene encodes OAH activity. OAH and OAH-like proteins form subclass of the isocitrate lyase/PEP mutase enzyme superfamily, which is ubiquitous present filamentous fungi. Analysis of function-specific residues using a superfamily-based approach revealed an active site serine as a possible sequence marker for OAH activity. We propose that presence of this serine in family members correlates with presence of OAH activity whereas its absence correlates with absence of OAH. This hypothesis was tested by carrying out a serine mutagenesis study with the OAH from the fungal oxalic acid producer Botrytis cinerea and the OAH active plant petal death protein as test systems.     

Morphology and citric acid production of Aspergillus niger PM 1

Summary Aspergillus niger PM 1 was grown in a tubular loop and a stirred tank bioreactor. Batch fermentations were performed under various agitation conditions and pH. Citric acid, oxalic acid, extracellular polysaccharides and proteins were assayed. The following morphological parameters were measured: mean perimeter of clumps, mean perimeter of the central core of clumps, mean length of filaments and mean diameter of filaments. Citric acid production and morphology in both reactors were dependent on agitation intensity and pH. The length of the filaments was shown to be the only parameter that could be related to citric acid production in both reactors: the shorter the filaments the more citric acid was produced. However, for the same amount of citric acid produced the morphology of the organism grown in the stirred tank differed considerably from that grown in the loop reactor.     

Bioleaching of spent refinery processing catalyst using Aspergillus niger with high-yield oxalic acid

A spent refinery processing catalyst was physically and chemically characterized, and subjected to one-step and two-step bioleaching processes using Aspergillus niger. During bioleaching of the spent catalysts of various particle sizes ("as received", 100-150 microm, <37 microm, and x =2.97 (average) microm) and pulp densities, the biomass dry weight and pH were determined. The corresponding leach liquor was analysed for excreted organic acids along with heavy metal values extracted from the catalyst. Chemical characterization of the spent catalyst confirmed the presence of heavy metal including Al (33.3%), Ni (6.09%) and Mo (13.72%). In general, the presence of the spent catalyst caused a decrease in the biomass yield and an increase in oxalic acid secretion by A. niger. The increase in oxalic acid secretion with a decrease in the catalyst particle size (up to <37 microm) led to corresponding increase in the extraction of metal values. The highest extraction of metal values from the spent catalyst (at 1% w/v pulp density and particle size <37 microm) were found to be 54.5% Al, 58.2% Ni and 82.3% Mo in 60 days of bioleaching. Oxalic acid secretion by A. niger in the presence of the spent catalyst was stimulated using 2-[N-Morpholino]ethanesulfonic acid (MES) buffer (pH 6), which resulted in comparable metal extraction (58% Al, 62.8% Ni and 78.9% Mo) in half the time required by the fungus in the absence of the buffer. Spent medium of A. niger grown in the absence and in the presence of MES buffer were found to leach almost similar amounts of Al and Ni, except Mo for which the spent medium of buffered culture was significantly more effective than the non-buffered culture. Overall, this study shows the possible use of bioleaching for the extraction of metal resources from spent catalysts. It also demonstrated the advantages of buffer-stimulated excretion of organic acids by A. niger in bioleaching of the spent catalyst.     

Production of oxalic acid by some fungi infected tubers

Oxalic acid (as oxalate) was detected in four tubers commonly used for food in Nigeria-Dioscorea rotundata (White yam), Solanum tuberosum (Irish potato), Ipomoea batatas (Sweet potato), and Manihot esculenta (cassava). Whereas healthy I. batata had the highest oxalic acid content, healthy M. esculenta contained the lowest. When all tubers were artifically inoculated with four fungi-Penicillium oxalicum CURIE and THOM, Aspergillus niger VAN TIEGH, A. flavus and A. tamarii KITA, there was an increase in oxalate content/g of tuber tissue. The greatest amount of oxalate was produced by P. oxalicum in D. rotundata tuber. Consistently higher amounts of oxalate were produced by the four fungi in infected sweet potato tuber than in any other tuber and consistently lower amounts of oxalate were produced by the four fungi in Irish potato tuber. Differences in the carbohydrate type present in the tubers and in the biosynthesis pathway are thought to be responsible for variation in the production of oxalate in the different tubers by the four fungi used.     

The ability of filamentous fungi to produce acids on indoor building materials

Sixty two filamentous fungi isolated from paint coatings, wallpaper, carton-gypsum board, and indoor air in buildings were screened for acid activity. It was found that 64.5% of strains produce acids on medium with bromo-cresol purple, where 18% of the strains were distinguished by very high acid activity (acid activity coefficient Q = 1.32–2.83), including the species:Aspergillus niger, Aspergillus versicolor, Penicillium expansum, Penicillium brevicom pactum, Penicillium chrysogenum, Cladosporium cladosporioides, Stachybotrys chartarum, Mucor globosus, Ulocladium chartarum andAlternaria alternata. Research indicated that filamentous fungi considerably decrease the pH of the medium when that medium containing building material. The greatest acid production and pH decrease of the medium was observed during the growth of filamentous fungi in a medium with mortar, while the production of acids was less in a medium with cartongypsum board, gypsum, and wallpaper. Filamentous fungi produced succinic, oxalic, malic and fumaric acids in the medium with indoor building materials. It was stated that the type of building material affects the spectrum and quantity of organic acids produced by filamentous fungi.     

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of Pi and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added 14CO2 was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this was oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.     

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger.

Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of Pi and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added 14CO2 was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this was oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.     

Oxalic acid production by citric acid-producing Aspergillus niger overexpressing the oxaloacetate hydrolase gene oahA

The filamentous fungus Aspergillus niger is used worldwide in the industrial production of citric acid. However, under specific cultivation conditions, citric acid-producing strains of A. niger accumulate oxalic acid as a by-product. Oxalic acid is used as a chelator, detergent, or tanning agent. Here, we sought to develop oxalic acid hyperproducers using A. niger as a host. To generate oxalic acid hyperproducers by metabolic engineering, transformants overexpressing the oahA gene, encoding oxaloacetate hydrolase (OAH; EC 3.7.1.1), were constructed in citric acid-producing A. niger WU-2223L as a host. The oxalic acid production capacity of this strain was examined by cultivation of EOAH-1 under conditions appropriate for oxalic acid production with 30 g/l glucose as a carbon source. Under all the cultivation conditions tested, the amount of oxalic acid produced by EOAH-1, a representative oahA-overexpressing transformant, exceeded that produced by A. niger WU-2223L. A. niger WU-2223L and EOAH-1 produced 15.6 and 28.9 g/l oxalic acid, respectively, during the 12-day cultivation period. The yield of oxalic acid for EOAH-1 was 64.2 % of the maximum theoretical yield. Our method for oxalic acid production gave the highest yield of any study reported to date. Therefore, we succeeded in generating oxalic acid hyperproducers by overexpressing a single gene, i.e., oahA, in citric acid-producing A. niger as a host.     

谷氨酸一钠制备新工艺研究

本文介绍了从谷氨酸发酵液中直接生产谷氨酸一钠（味精）的现实性与可能性;探讨了新工艺的最佳条件。     

Bioleaching of spent fluid catalytic cracking catalyst using Aspergillus niger

The use of the fungus Aspergillus niger for the bioleaching of heavy metals from spent catalyst was investigated, with fluid catalytic cracking (FCC) catalyst as a model. Bioleaching was examined in batch cultures with the spent catalysts at various pulp densities (1-12%). Chemical leaching was also performed using mineral acids (sulphuric and nitric acids) and organic acids (citric, oxalic and gluconic acids), as well as a mixture of organic acids at the same concentrations as that biogenically produced. It was shown that bioleaching realised higher metal extraction than chemical leaching, with A. niger mobilizing Ni (9%), Fe (23%), Al (30%), V (36%) and Sb (64%) at 1% pulp density. Extraction efficiency generally decreased with increased pulp density. Compared with abiotic controls, bioleaching gave rise to higher metal extractions than leaching using fresh medium and cell-free spent medium. pH decreased during bioleaching, but remained relatively constant in both leaching using fresh medium and cell-free spent medium, thus indicating that the fungus played a role in effecting metal extraction from the spent catalyst.     

Influence of the glycolytic rate on production of citric acid and oxalic acid by Aspergillus niger in solid state fermentation

A study was performed to understand the physiology and biochemical mechanism of citric acid accumulation during solid state fermentation of sweet potato using Aspergillus niger Yang No.2. A low citrate-producing mutant was isolated followed by a comparative study of the fermentation process and selected physiological and biochemical parameters. In contrast with the parent strain, the mutant strain displayed lower concentrations, yields and production rates of citric acid, accompanied by higher concentrations, yields and production rates of oxalic acid. In addition, the mutant utilized starch at a lower rate although higher concentrations of free glucose accumulated in the cultures. Biochemical analyses revealed lower rates of glucose uptake and hexokinase activity of the mutant strain in comparison with the parent strain. It is proposed that, in common with submerged fermentation, over-production of citric acid in solid state fermentation is related to an increased glucose flux through glycolysis. At low glucose fluxes, oxalic acid is accumulated.     

植酸酶产生菌黑曲霉N14的诱变选育及其基因分析

以植酸酶产生菌黑曲霉03214为出发菌株,经紫外线和亚硝基胍诱变,获得了产酶活性较出发菌株提高了22．3％,达422IU／ml发酵液的突变菌株黑曲霉N14,其最适pH值为2．5,最适温度为50℃。通过对黑曲霉N14植酸酶phyA基因进行PCR扩增,获得了一条长约1．5kb的特异性产物。以pMD18-T为载体,构建了含有目的基因片段的重组质粒。DNA序列测定表明,目的基因片段含有植酸酶phyA基因的完整序列(GenBank Accession：AY426977),phyA基因全长1506bp,其中包含一段长102bp的内含子,编码467个氨基酸,有10个潜在的糖基化位点,5’端有一编码19个氨基酸的信号肽序列。实验结果为植酸酶基因工程菌的构建奠定了基础。     

Rock phosphate solubilization by abiotic and fungal-produced oxalic acid: reaction parameters and bioleaching potential

Oxalic acid-producing fungi play an important role in biogeochemical transformations of rocks and minerals and possess biotechnological potential for extraction of valuable elements from primary or waste ores and other solid matrices. This research investigates the extraction of phosphate from rock phosphate (RP) by oxalic acid. Reaction parameters were derived using pure oxalic acid solutions to solubilize RP. It was found that the oxalic acid concentration was the main factor driving reaction kinetics. Excess oxalic acid could retard the reaction due to calcium oxalate encrustation on RP surfaces. However, complete P extraction was reached at stoichiometric proportions of apatite and oxalic acid. This reaction reached completion after 168 h, although most of the P (up to 75%) was released in less than 1 h. Most of the Ca released from the apatite formed sparingly soluble calcium oxalate minerals, with a predominance of whewellite over weddellite. Bioleaching of RP employing biomass-free spent culture filtrates containing oxalic acid (100 mM) produced by Aspergillus niger extracted ~ 74% of the P contained in the RP. These findings contribute to a better understanding of the reaction between apatite and oxalic acid and provide insights for potential applications of this process for biotechnological production of phosphate fertilizer.     

高效溶磷菌的分离、筛选及在土壤中溶磷有效性的研究

从采自全国各地130个土样中.分离出1000余菌株,从中筛选出1株代号为Ap-2号的高效溶磷菌.经鉴定为黑曲霉Aspergillus niger.对该菌株的产酸特性以及接种土壤后的溶磷有效性进行了研究.试验表明,该菌株发酵过程产草酸、柠檬酸等多种有机酸,使土壤速效磷含量增加141.94%.其溶磷作用,与土壤pH含水量有密切关系,对温度要求不严,与接种量是一定正相关.并可与磷酸铵、三料、尿素、硫酸铵等化肥混合使用.     

Production of citric and oxalic acids and solubilization of calcium phosphate by Penicillium bilaii.

An isolate of Penicillium bilaii previously reported to solubilize mineral phosphates and enhance plant uptake of phosphate was studied. Using agar media with calcium phosphate and the pH indicator alizarin red S, the influence of the medium composition on phosphate solubility and medium acidification was recorded. The major acidic metabolites produced by P. bilaii in a sucrose nitrate liquid medium were found to be oxalic acid and citric acid. Citric acid production was promoted under nitrogen-limited conditions, while oxalic acid production was promoted under carbon-limited conditions. Citric acid was produced in both growth and stationary phases, but oxalic acid production occurred only in stationary phase. When submerged cultures which normally produce acid were induced to sporulate, the culture medium shifted toward alkaline rather than acid reaction with growth.     

Heterologous gene expression in Aspergillus niger: a glucoamylase-porcine pancreatic prophospholipase A2 fusion protein is secreted and processed to yield mature enzyme.

The cDNA gene encoding porcine pancreatic prophospholipase A2 (proPLA2) was cloned into an Aspergillus niger expression vector downstream of the glucoamylase (glaA) gene promoter region. When this construct was transformed into A. niger, no detectable PLA2 was produced. Evidence was obtained showing that the PLA2 gene was transcribed and that PLA2 is extremely susceptible to both intracellular and extracellular proteases of A. niger, thus indicating that translation products would be rapidly degraded. By fusing the proPLA2-encoding sequence to the entire glaA gene, secreted yields of PLA2 up to 10 micrograms/ml were obtained from a transformed protease-deficient strain of A. niger. PLA2 was secreted in young cultures as a fusion protein, but in older cultures, it was processed from the glucoamylase carrier protein. Secreted PLA2 was shown to be enzymatically active and to have the correct N-terminal amino acid (aa) sequence, although another form of processed PLA2 was also produced. This form included two aa of the proregion from PLA2. The potential for improving yields of secreted heterologous proteins from A. niger still further is discussed.