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Application of methanol and sweet potato vine hydrolysate as enhancers of citric acid production by Aspergillus niger.

ABSTRACT: Agricultural waste is as an alternative low-cost carbon source or beneficial additives which catch most people's eyes. In addition, methanol and sweet potato vine hydrolysate (SVH) have been reported as the efficient enhancers of fermentation according to some reports. The objective of the present study was to confirm SVH as an efficient additive in CA production and explore the synergistic effects of methanol and SVH in fermentation reactions.The optimal fermentation conditions resulted in a maximum citric acid concentration of 3.729 g/L. The final citric acid concentration under the optimized conditions was increased by 3.6-fold over the original conditions, 0.49-fold over the optimized conditions without methanol, and 1.8-fold over the optimized conditions in the absence of SVH. Kinetic analysis showed that Qp, Yp/s, and Yx/s in the optimized systems were significantly improved compared with those obtained in the absence of methanol or SVH. Further, scanning electron microscopy (SEM) revealed that methanol stress promoted the formation of conidiophores, while SVH could neutralize the effect and prolong Aspergillus niger vegetative growth. Cell viability analysis also showed that SVH might eliminate the harmful effects of methanol and enhance cell membrane integrity.SVH was a superior additive for organic acid fermentation, and the combination of methanol and SVH displayed a significant synergistic effect. The research provides a preliminary theoretical basis for SVH practical application in the fermentation industry.

SUBMITTER: Yu D

PROVIDER: S-EPMC5532408 | biostudies-other | 2017

REPOSITORIES: biostudies-other

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Integration of Aspergillus niger transcriptomic profile with metabolic model identifies potential targets to optimise citric acid production from lignocellulosic hydrolysate.

Project description:BackgroundCitric acid is typically produced industrially by Aspergillus niger-mediated fermentation of a sucrose-based feedstock, such as molasses. The fungus Aspergillus niger has the potential to utilise lignocellulosic biomass, such as bagasse, for industrial-scale citric acid production, but realising this potential requires strain optimisation. Systems biology can accelerate strain engineering by systematic target identification, facilitated by methods for the integration of omics data into a high-quality metabolic model. In this work, we perform transcriptomic analysis to determine the temporal expression changes during fermentation of bagasse hydrolysate and develop an evolutionary algorithm to integrate the transcriptomic data with the available metabolic model to identify potential targets for strain engineering.ResultsThe novel integrated procedure matures our understanding of suboptimal citric acid production and reveals potential targets for strain engineering, including targets consistent with the literature such as the up-regulation of citrate export and pyruvate carboxylase as well as novel targets such as the down-regulation of inorganic diphosphatase.ConclusionsIn this study, we demonstrate the production of citric acid from lignocellulosic hydrolysate and show how transcriptomic data across multiple timepoints can be coupled with evolutionary and metabolic modelling to identify potential targets for further engineering to maximise productivity from a chosen feedstock. The in silico strategies employed in this study can be applied to other biotechnological goals, assisting efforts to harness the potential of microorganisms for bio-based production of valuable chemicals.

| S-EPMC8756645 | biostudies-literature

Systems metabolic engineering for citric acid production by Aspergillus niger in the post-genomic era.

Project description:Citric acid is the world's largest consumed organic acid and is widely used in beverage, food and pharmaceutical industries. Aspergillus niger is the main industrial workhorse for citric acid production. Since the release of the genome sequence, extensive multi-omic data are being rapidly obtained, which greatly boost our understanding of the citric acid accumulation mechanism in A. niger to a molecular and system level. Most recently, the rapid development of CRISPR/Cas9 system facilitates highly efficient genome-scale genetic perturbation in A. niger. In this review, we summarize the impact of systems biology on the citric acid molecular regulatory mechanisms, the advances in metabolic engineering strategies for enhancing citric acid production and discuss the development and application of CRISPR/Cas9 systems for genome editing in A. niger. We believe that future systems metabolic engineering efforts will redesign and engineer A. niger as a highly optimized cell factory for industrial citric acid production.

| S-EPMC6362574 | biostudies-literature

NADP-specific isocitrate dehydrogenase from the citric acid-accumulating fungus Aspergillus niger.

Project description:NADP-specific isocitrate dehydrogenase [threo-DS-isocitrate: NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] was purified 200-300-fold from the citric acid-accumulating fungus Aspergillus niger. The enzyme consists of a single polypeptide chain with a molecular mass of 60 +/- 4 kDa and has a pI of 5.9 +/- 0.2. Only a single enzyme protein was found, although the enzyme appears to occur both in the mitochondrion and in the cytoplasm. Growth on organic acids as carbon sources or on NO3- as nitrogen source led to increased activities, whereas the presence of amino acids led to lower activities. The enzyme exhibits hyperbolic kinetics with respect to its substrates isocitrate and NADP+. Mn2+ and Mg2+ are obligatory for enzyme activity. The enzyme is inhibited by its products alpha-oxoglutarate and NADPH. Among various metabolites, ATP and citrate appear to inhibit the enzyme at a concentration considered to occur intracellularly. In both cases, however, the mechanism is a removal of the metal ion cofactor from the substrates. It is concluded that under physiological conditions, where the Mg2+ content is around 10 mM, the observed inhibition by ATP or citrate is of poor regulatory significance.

| S-EPMC1146875 | biostudies-other

Biotechnological valorization of cashew apple juice for the production of citric acid by a local strain of Aspergillus niger LCFS 5.

Project description:BackgroundThis work investigates the production of citric acid from cashew apple juice, an abundant waste in the processing of cashew, using a local strain of Aspergillus niger and the application of the citric acid as a coagulant for the production of soy cheese. Fungal isolates were obtained from a cashew plantation in Ogbomoso, Nigeria, using potato dextrose agar. Further screening was undertaken to determine the qualitative strength of acid production by the fungi on Czapek-Dox agar supplemented with bromocresol green, with the development of yellow zone taken as an indication of citric acid production. Thereafter, the best producing strain was cultivated in a cashew apple juice medium.ResultsOut of 150 fungal isolates generated from the cashew plantation, 92 (61.3%), 44 (29.3%) and 14 (9.3%) were obtained from cashew fruits, soil and cashew tree surfaces, respectively. Different strains of fungi isolated include Aspergillus niger, A. flavus, A. foetidus, A. heteromorphus, A. nidulans and A. viridinutans. The isolates produced yellow zonation of 0.4-5.5 cm on modified Czapek-Dox agar; the highest was observed for a strain of A. niger LCFS 5, which was identified using molecular tools. In the formulated cashew apple juice medium, the citric acid yield of LCFS 5 ranged 16.0-92.8 g/l with the peak obtained on the 10th day of fermentation. The citric acid produced was recovered using the double precipitation method with Ca(OH)2 and H2SO4 having ≈ 70% purity of citric acid on HPLC. The citric acid acted as a coagulant to produce soy cheese with 66.67% acceptability by panelists.ConclusionThis work has extended the frontiers of valorization of cashew waste by a strain of A. niger to produce citric acid in high yield, with potential application in food industries.

| S-EPMC8448800 | biostudies-literature

Phytase production by Aspergillus niger NCIM 563 for a novel application to degrade organophosphorus pesticides.

Project description:The production of phytase using Aspergillus niger NCIM 563 under submerged fermentation conditions was studied using protein rich chickpea flour as substrate. Employing a hybrid statistical media optimization strategy of Plackett-Burman and Box-Behnken experimental designs in shake-flasks gave an increased phytase activity from an initial 66 IU/mL in 216 h to 160 IU/mL in a reduced time of 132 h. Productivity, thus increased by 3.97 times from 7.3 to 29 IU/mL/day. Using the optimized media, the production was successfully scaled-up further and improved up to 164 IU/mL in 96 h by studies carried out employing 2 and 10-L fermenters. The enzyme supernatant was recovered using centrifugal separation of biomass and the stability of the produced phytase was tested for animal feed applications under gastric conditions. In vitro degradation studies of water soluble monocrotophos, methyl parathion and water insoluble chlorpyrifos, pesticides used extensively in agriculture was carried out. It was observed by HPLC analysis that phytase could degrade 72% of chlorpyrifos at pH 7.0, 35 °C. Comparable results were obtained with monocrotophos and methyl parathion. With chlorpyrifos at higher temperature 50 °C as much as 91% degradation could be obtained. The degradation of chlorpyrifos was further validated by spraying phytase on harvested green chilli (Capsicum annuum L) under normal conditions of pH 7.0, 35 °C and the degradation products obtained analyzed by LCMS. Thus, the present study brings out a potentially novel application of phytase for biodegradation of organophosphorus pesticides.

| S-EPMC5359262 | biostudies-literature

Engineering a Phosphoketolase Pathway to Supplement Cytosolic Acetyl-CoA in <i>Aspergillus niger</i> Enables a Significant Increase in Citric Acid Production.

Project description:Citric acid is widely used in the food, chemical and pharmaceutical industries. Aspergillus niger is the workhorse used for citric acid production in industry. A canonical citrate biosynthesis that occurred in mitochondria was well established; however, some research suggested that the cytosolic citrate biosynthesis pathway may play a role in this chemical production. Here, the roles of cytosolic phosphoketolase (PK), acetate kinase (ACK) and acetyl-CoA synthetase (ACS) in citrate biosynthesis were investigated by gene deletion and complementation in A. niger. The results indicated that PK, ACK and ACS were important for cytosolic acetyl-CoA accumulation and had significant effects on citric acid biosynthesis. Subsequently, the functions of variant PKs and phosphotransacetylase (PTA) were evaluated, and their efficiencies were determined. Finally, an efficient PK-PTA pathway was reconstructed in A. niger S469 with Ca-PK from Clostridium acetobutylicum and Ts-PTA from Thermoanaerobacterium saccharolyticum. The resultant strain showed an increase of 96.4% and 88% in the citrate titer and yield, respectively, compared with the parent strain in the bioreactor fermentation. These findings indicate that the cytosolic citrate biosynthesis pathway is important for citric acid biosynthesis, and increasing the cytosolic acetyl-CoA level can significantly enhance citric acid production.

| S-EPMC10219267 | biostudies-literature

Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88.

Project description:The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi.

| S-EPMC3106321 | biostudies-literature

Improving citric acid production of an industrial Aspergillus niger CGMCC 10142: identification and overexpression of a high-affinity glucose transporter with different promoters.

Project description:BackgroundGlucose transporters play an important role in the fermentation of citric acid. In this study, a high-affinity glucose transporter (HGT1) was identified and overexpressed in the industrial strain A. niger CGMCC 10142. HGT1-overexpressing strains using the PglaA and Paox1 promoters were constructed to verify the glucose transporter functions.ResultAs hypothesized, the HGT1-overexpressing strains showed higher citric acid production and lower residual sugar contents. The best-performing strain A. niger 20-15 exhibited a reduction of the total sugar content and residual reducing sugars by 16.5 and 44.7%, while the final citric acid production was significantly increased to 174.1 g/L, representing a 7.3% increase compared to A. niger CGMCC 10142. Measurement of the mRNA expression levels of relevant genes at different time-points during the fermentation indicated that in addition to HGT1, citrate synthase and glucokinase were also expressed at higher levels in the overexpression strains.ConclusionThe results indicate that HGT1 overexpression resolved the metabolic bottleneck caused by insufficient sugar transport and thereby improved the sugar utilization rate. This study demonstrates the usefulness of the high-affinity glucose transporter HGT1 for improving the citric acid fermentation process of Aspergillus niger CGMCC 10142.

| S-EPMC8394697 | biostudies-literature

Isolation, identification, and characterization of an Aspergillus niger bioflocculant-producing strain using potato starch wastewater as nutrilite and its application.

Project description:A bioflocculant (MBFA18) was produced by Aspergillus niger (A18) using potato starch wastewater (PSW) as nutrients. The cultivation processes and flocculating treatment for PSW purification were systematically studied. The flocculating rate of the MBFA 18 achieved 90.06% (kaolin clay) under the optimal cultivation condition (PSW with 5950 mg/L COD, 20 g/L glucose, 0.2 g/L urea and without phosphorus source addition and pH adjustment). Furthermore, effects of flocculant dosage, initial pH, coagulant aid (CaCl2) addition and sedimentation time on the PSW treatment were discussed and studied in detail. The optimum flocculation treatment conditions were determined according to the treatment efficiency, cost and flocculation conditions. During the PSW treatment, 2 mL/L bioflocculant (1.89 g/L) dosage and 0.5 mol/L coagulant aid addition were applied without pH adjustment and 91.15% COD and 60.22% turbidity removal rate could be achieved within 20 min. The comparative study between the bioflocculant and conventional chemical flocculants showed excellent flocculating efficiency of MBFA 18 with lower cost (4.7 yuan/t), which indicated that the bioflocculant MBFA 18 produced in PSW substrate has a great potential to be an alternative flocculant in PSW treatment.

| S-EPMC5755778 | biostudies-literature

Evaluation of <i>Aspergillus niger</i> Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production.

Project description:Aspergillus niger is an important industrial workhorse for the biomanufacturing of organic acids, proteins, etc. Well-controlled genetic regulatory elements, including promoters, are vital for strain engineering, but available strong promoters for A. niger are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on mCherry, pyrG, CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in A. niger. The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter PgpdAg showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter PgpdAd from A. nidulans. Six predicted conserved motifs, including the gpdA-box, were verified to be essential for the PgpdAg activity. To demonstrate its application, the promoter PgpdAg was used for enhancing the expression of citrate exporter cexA in a citric acid-producing isolate D353.8. Compared with the cexA controlled by PgpdAd, the transcription level of the cexA gene driven by PgpdAg increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following cexA overexpression, several genes involved in carbohydrate transport and metabolism were synergically upregulated, resulting in up to a 2.48-fold increase in citric acid titer compared with that of the parent strain. This study provides an intuitive workflow to speed up the quantitative evaluation of A. niger promoters and strong constitutive promoters for fungal cell factory construction and strain engineering.

| S-EPMC9224621 | biostudies-literature

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