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

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.

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

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.760500 ◽

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.

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Production of lactic and acetic acids during fermentation of milk fortified with kiwi juice using Saccharomyces boulardii and lactobacilli

Tropical Journal of Pharmaceutical Research ◽

10.4314/tjpr.v18i4.1 ◽

2021 ◽

Vol 18 (4) ◽

pp. 681-687

Author(s):

Ahmed Hassan Mousa ◽

Gang Wang ◽

Hao Zhang

Keyword(s):

Mass Spectrometry ◽

Gas Chromatography ◽

Acetic Acid ◽

Acid Production ◽

Saccharomyces Boulardii ◽

Gas Chromatography Mass Spectrometry ◽

Acid Yield ◽

Marginal Production ◽

Skimmed Milk ◽

Acetic Acids

Purpose: To investigate the synergistic effect of Saccharomyces boulardii and lactobacilli on lactic and acetic acids produced during fermentation of milk fortified with kiwi juice, relative to fermentation of unfortified milk. Methods: Skimmed milk was fortified with kiwi juice (4 % v/v) and fermented for 12 h at 37 °C by a combination of S. boulardii and lactobacilli strains. Lactic and acetic acids were determined using gas chromatography-mass spectrometry (GS-MS). Results: The presence of kiwi juice in the milk stimulated the production of lactic (1.35 g/100g) and acetic (0.29 mg/g) by S. boulardii in the absence of lactobacilli. When S. boulardii was inoculated with Lb. casei 20975, the production of lactic acid and acetic acid increased to 2.36 g/100 g and 0.71 mg/g, respectively. Furthermore, acid production increased when Lb. plantarum RS (35-11), Lb. casei LCS, and Lb. plantarum JXJ (6 - 12) were inoculated into milk free of kiwi juice in which S. boulardii was grown. Saccharomyces boulardii resulted in marginal production of acids by Lb. fermentum F9. Conclusion: These results show that acid production is positively affected by some lactobacilli strains in the milk whether fortified with kiwi juice or free of this juice. However, fermentation of these formulations for a period longer than 6 h may result in losses in acid yield.

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l-Lactic Acid Production Using Engineered Saccharomyces cerevisiae with Improved Organic Acid Tolerance

Journal of Fungi ◽

10.3390/jof7110928 ◽

2021 ◽

Vol 7 (11) ◽

pp. 928

Author(s):

Byeong-Kwan Jang ◽

Yebin Ju ◽

Deokyeol Jeong ◽

Sung-Keun Jung ◽

Chang-Kil Kim ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Acetic Acid ◽

Lactic Acid ◽

Acid Production ◽

Lactic Acid Production ◽

Acid Tolerance ◽

Production Costs ◽

High Concentration ◽

Adaptive Laboratory Evolution ◽

Engineered Saccharomyces Cerevisiae

Lactic acid is mainly used to produce bio-based, bio-degradable polylactic acid. For industrial production of lactic acid, engineered Saccharomyces cerevisiae can be used. To avoid cellular toxicity caused by lactic acid accumulation, pH-neutralizing agents are used, leading to increased production costs. In this study, lactic acid-producing S. cerevisiae BK01 was developed with improved lactic acid tolerance through adaptive laboratory evolution (ALE) on 8% lactic acid. The genetic basis of BK01 could not be determined, suggesting complex mechanisms associated with lactic acid tolerance. However, BK01 had distinctive metabolomic traits clearly separated from the parental strain, and lactic acid production was improved by 17% (from 102 g/L to 119 g/L). To the best of our knowledge, this is the highest lactic acid titer produced by engineered S. cerevisiae without the use of pH neutralizers. Moreover, cellulosic lactic acid production by BK01 was demonstrated using acetate-rich buckwheat husk hydrolysates. Particularly, BK01 revealed improved tolerance against acetic acid of the hydrolysates, a major fermentation inhibitor of lignocellulosic biomass. In short, ALE with a high concentration of lactic acid improved lactic acid production as well as acetic acid tolerance of BK01, suggesting a potential for economically viable cellulosic lactic acid production.

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

Fermentation ◽

10.3390/fermentation8010026 ◽

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.

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