scholarly journals Immobilization of Aspergillus oryzae DSM 1863 for l-Malic Acid Production

Fermentation ◽  
2022 ◽  
Vol 8 (1) ◽  
pp. 26
Author(s):  
Aline Kövilein ◽  
Vera Aschmann ◽  
Silja Hohmann ◽  
Katrin Ochsenreither
Keyword(s):  
Aspergillus Oryzae ◽  
Malic Acid ◽  
Acid Production ◽  
Carbon Sources ◽  
Cell Immobilization ◽  
Batch Processes ◽  
Natural Polymers ◽  
Biomass Retention ◽  
Shake Flasks ◽  
Acetate Medium

Whole-cell immobilization by entrapment in natural polymers can be a tool for morphological control and facilitate biomass retention. In this study, the possibility of immobilizing the filamentous fungus Aspergillus oryzae for l-malic acid production was evaluated with the two carbon sources acetate and glucose. A. oryzae conidia were entrapped in alginate, agar, and κ-carrageenan and production was monitored in batch processes in shake flasks and 2.5-L bioreactors. With glucose, the malic acid concentration after 144 h of cultivation using immobilized particles was mostly similar to the control with free biomass. In acetate medium, production with immobilized conidia of A. oryzae in shake flasks was delayed and titers were generally lower compared to cultures with free mycelium. While all immobilization matrices were stable in glucose medium, disintegration of bead material and biomass detachment in acetate medium was observed in later stages of the fermentation. Still, immobilization proved advantageous in bioreactor cultivations with acetate and resulted in increased malic acid titers. This study is the first to evaluate immobilization of A. oryzae for malic acid production and describes the potential but also challenges regarding the application of different matrices in glucose and acetate media.

scholarly journals Enhanced l-Malic Acid Production by Aspergillus oryzae DSM 1863 Using Repeated-Batch Cultivation

2022 ◽  
Vol 9 ◽  
Author(s):  
Vanessa Schmitt ◽  
Laura Derenbach ◽  
Katrin Ochsenreither
Keyword(s):  
Calcium Carbonate ◽  
Aspergillus Oryzae ◽  
Malic Acid ◽  
Acid Production ◽  
Type Strain ◽  
Wild Type Strain ◽  
Batch Process ◽  
Batch Cultivation ◽  
Wild Type ◽  
Repeated Batch

l-Malic acid is a C4-dicarboxylic acid and a potential key building block for a bio-based economy. At present, malic acid is synthesized petrochemically and its major market is the food and beverages industry. In future, malic acid might also serve as a building block for biopolymers or even replace the commodity chemical maleic anhydride. For a sustainable production of l-malic acid from renewable resources, the microbial synthesis by the mold Aspergillus oryzae is one possible route. As CO2 fixation is involved in the biosynthesis, high yields are possible, and at the same time greenhouse gases can be reduced. In order to enhance the production potential of the wild-type strain Aspergillus oryzae DSM 1863, process characteristics were studied in shake flasks, comparing batch, fed-batch, and repeated-batch cultivations. In the batch process, a prolonged cultivation time led to malic acid consumption. Keeping carbon source concentration on a high level by pulsed feeding could prolong cell viability and cultivation time, however, did not result in significant higher product levels. In contrast, continuous malic acid production could be achieved over six exchange cycles and a total fermentation time of 19 days in repeated-batch cultivations. Up to 178 g/L l-malic acid was produced. The maximum productivity (0.90 ± 0.05 g/L/h) achieved in the repeated-batch cultivation had more than doubled than that achieved in the batch process and also the average productivity (0.42 ± 0.03 g/L/h for five exchange cycles and 16 days) was increased considerably. Further repeated-batch experiments confirmed a positive effect of regular calcium carbonate additions on pH stability and malic acid synthesis. Besides calcium carbonate, nitrogen supplementation proved to be essential for the prolonged malic acid production in repeated-batch. As prolonged malic acid production was only observed in cultivations with product removal, product inhibition seems to be the major limiting factor for malic acid production by the wild-type strain. This study provides a systematic comparison of different process strategies under consideration of major influencing factors and thereby delivers important insights into natural l-malic acid production.

scholarly journals Acetate as substrate for l-malic acid production with Aspergillus oryzae DSM 1863

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Aline Kövilein ◽  
Julia Umpfenbach ◽  
Katrin Ochsenreither
Keyword(s):  
Acetic Acid ◽  
Carbon Source ◽  
Aspergillus Oryzae ◽  
Malic Acid ◽  
Acid Production ◽  
Morphological Changes ◽  
Production Costs ◽  
High Tolerance ◽  
Acid Yield ◽  
Acetate Concentration

Abstract Background Microbial malic acid production is currently not able to compete economically with well-established chemical processes using fossil resources. The utilization of inexpensive biomass-based substrates containing acetate could decrease production costs and promote the development of microbial processes. Acetate is a by-product in lignocellulosic hydrolysates and fast pyrolysis products or can be synthesized by acetogens during syngas fermentation. For the fermentation of these substrates, a robust microorganism with a high tolerance for biomass-derived inhibitors is required. Aspergillus oryzae is a suitable candidate due to its high tolerance and broad substrate spectrum. To pave the path towards microbial malic acid production, the potential of acetate as a carbon source for A. oryzae is evaluated in this study. Results A broad acetate concentration range was tested both for growth and malic acid production with A. oryzae. Dry biomass concentration was highest for acetic acid concentrations of 40–55 g/L reaching values of about 1.1 g/L within 48 h. Morphological changes were observed depending on the acetate concentration, yielding a pellet-like morphology with low and a filamentous structure with high substrate concentrations. For malic acid production, 45 g/L acetic acid was ideal, resulting in a product concentration of 8.44 ± 0.42 g/L after 192 h. The addition of 5–15 g/L glucose to acetate medium proved beneficial by lowering the time point of maximum productivity and increasing malic acid yield. The side product spectrum of cultures with acetate, glucose, and cultures containing both substrates was compared, showing differences especially in the amount of oxalic, succinic, and citric acid produced. Furthermore, the presence of CaCO3, a pH regulator used for malate production with glucose, was found to be crucial also for malic acid production with acetate. Conclusions This study evaluates relevant aspects of malic acid production with A. oryzae using acetate as carbon source and demonstrates that it is a suitable substrate for biomass formation and acid synthesis. The insights provided here will be useful to further microbial malic acid production using renewable substrates.

scholarly journals Key Process Conditions for Production of C4 Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae Strain

2009 ◽  
Vol 76 (3) ◽  
pp. 744-750 ◽  
Author(s):  
Rintze M. Zelle ◽  
Erik de Hulster ◽  
Wendy Kloezen ◽  
Jack T. Pronk ◽  
Antonius J. A. van Maris
Keyword(s):  
Carbon Dioxide ◽  
Saccharomyces Cerevisiae ◽  
Process Parameters ◽  
Malic Acid ◽  
Acid Production ◽  
Shake Flask ◽  
Simultaneous Optimization ◽  
Process Conditions ◽  
Recent Effort ◽  
Shake Flasks

ABSTRACT A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter−1 of malate at a yield of 0.42 mol (mol glucose)−1 in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO3, was required for efficient C4 dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO3 dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose)−1, whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered S. cerevisiae strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO2, calcium, and O2). Under optimized conditions, a malate yield of 0.48 ± 0.01 mol (mol glucose)−1 was obtained in bioreactors, a 19% increase over yields in shake flask experiments.

Fungal biotransformation of crude glycerol into malic acid

2015 ◽  
Vol 70 (5-6) ◽  
pp. 165-167 ◽  
Author(s):  
Thomas P. West
Keyword(s):  
Organic Acid ◽  
Biomass Production ◽  
Malic Acid ◽  
Acid Production ◽  
Fungal Biomass ◽  
Acid Level ◽  
Crude Glycerol

Abstract Malic acid production from the biodiesel coproduct crude glycerol by Aspergillus niger ATCC 9142, ATCC 10577 and ATCC 12846 was observed to occur with the highest malic acid level acid being produced by A. niger ATCC 12846. Fungal biomass production from crude glycerol was similar, but ATCC 10577 produced the highest biomass. Fungal biotransformation of crude glycerol into the commercially valuable organic acid malic acid appeared feasible.

Overexpression of a novel gene Aokap2 affects the growth and kojic acid production in Aspergillus oryzae

2022 ◽  
Author(s):  
Yuzhen Li ◽  
Huanxin Zhang ◽  
Ziming Chen ◽  
Junxia Fan ◽  
Tianming Chen ◽  
...  
Keyword(s):  
Aspergillus Oryzae ◽  
Acid Production ◽  
Kojic Acid ◽  
Novel Gene ◽  
Kojic Acid Production

Characteristics of the high malic acid production mechanism in Saccharomyces cerevisiae sake yeast strain No. 28

2012 ◽  
Vol 114 (3) ◽  
pp. 281-285 ◽  
Author(s):  
Shunichi Nakayama ◽  
Ken Tabata ◽  
Takahiro Oba ◽  
Kenichi Kusumoto ◽  
Shinji Mitsuiki ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Malic Acid ◽  
Yeast Strain ◽  
Acid Production ◽  
Production Mechanism

scholarly journals Investigation of Malic Acid Production in Aspergillus oryzae under Nitrogen Starvation Conditions

2013 ◽  
Vol 79 (19) ◽  
pp. 6050-6058 ◽  
Author(s):  
Christoph Knuf ◽  
Intawat Nookaew ◽  
Stephen H. Brown ◽  
Michael McCulloch ◽  
Alan Berry ◽  
...  
Keyword(s):  
Malic Acid ◽  
Acid Production ◽  
Large Scale ◽  
High Capacity ◽  
Nitrogen Sources ◽  
Building Blocks ◽  
Close Relative ◽  
Biotechnological Production ◽  
Content Type ◽  
The One

ABSTRACTMalic acid has great potential for replacing petrochemical building blocks in the future. For this application, high yields, rates, and titers are essential in order to sustain a viable biotechnological production process. Natural high-capacity malic acid producers like the malic acid producerAspergillus flavushave so far been disqualified because of special growth requirements or the production of mycotoxins. AsA. oryzaeis a very close relative or even an ecotype ofA. flavus, it is likely that its high malic acid production capabilities with a generally regarded as safe (GRAS) status may be combined with already existing large-scale fermentation experience. In order to verify the malic acid production potential, two wild-type strains, NRRL3485 and NRRL3488, were compared in shake flasks. As NRRL3488 showed a volumetric production rate twice as high as that of NRRL3485, this strain was selected for further investigation of the influence of two different nitrogen sources on malic acid secretion. The cultivation in lab-scale fermentors resulted in a higher final titer, 30.27 ± 1.05 g liter−1, using peptone than the one of 22.27 ± 0.46 g liter−1obtained when ammonium was used. Through transcriptome analysis, a binding site similar to the one of theSaccharomyces cerevisiaeyeast transcription factor Msn2/4 was identified in the upstream regions of glycolytic genes and the cytosolic malic acid production pathway from pyruvate via oxaloacetate to malate, which suggests that malic acid production is a stress response. Furthermore, the pyruvate carboxylase reaction was identified as a target for metabolic engineering, after it was confirmed to be transcriptionally regulated through the correlation of intracellular fluxes and transcriptional changes.

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