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

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.

Biodegradation of a Complex Phenolic Industrial Stream by Bacterial Strains Isolated from Industrial Wastewaters

Molecular and metabolomic tools were used to design and understand the biodegradation of phenolic compounds in real industrial streams. Bacterial species were isolated from an industrial wastewater treatment plant of a phenol production factory and identified using molecular techniques. Next, the biodegradation potential of the most promising strains was analyzed in the presence of a phenolic industrial by-product containing phenol, alfa-methylstyrene, acetophenone, 2-cumylphenol, and 4-cumylphenol. A bacterial consortium comprising Pseudomonas and Alcaligenes species was assessed for its ability to degrade phenolic compounds from the phenolic industrial stream (PS). The consortium adapted itself to the increasing levels of phenolic compounds, roughly up to 1750 ppm of PS; thus, becoming resistant to them. In addition, the consortium exhibited the ability to grow in the presence of PS in repeated batch mode processes. Results from untargeted metabolomic analysis of the culture medium in the presence of PS suggested that bacteria transformed the toxic phenolic compounds into less harmful molecules as a survival mechanism. Overall, the study demonstrates the usefulness of massive sequencing and metabolomic tools in constructing bacterial consortia that can efficiently biodegrade complex PS. Furthermore, it improves our understanding of their biodegradation capabilities.

Photocatalytic Decomposition of Organic Dyes Using a Rotating Cylinder System

In this study, a rotating cylinder system was used in the photocatalytic decomposition of organic dyes in aqueous medium for water purification. To this end, the titania nanoparticle dispersion was mixed with an organic dye solution under a rotating inner cylinder at controlled speed. The rate constant was adjusted by changing the speed of rotation to determine the optimal circulating velocity. Since nanoparticle dispersion is a secondary contaminant after wastewater treatment, the titania paste was deposited on the inner surface of the stationary outer cylinder to form a photocatalytic film. During repeated batch-mode operation, the deactivation of the deposited film was analyzed by measuring the rate constant as a function of time. Continuous operation was also used to remove organic dye in the water to study factors affecting the removal efficiency of methylene blue. Higher rotating velocity and slow feed rate facilitated the removal of contaminants via desorption of adsorbed dyes with adequate retention time.

Production of Docosahexaenoic Acid, DHA using Different Modes of Cultivation by Aurantiochytrium sp. SW1

Thraustochytrids, such as members of the genus Aurantiochytrium, are rich in docosahexaenoic acid (DHA, C22:6n-3) and represent a promising source of omega-3 fatty acids which plays a vital role in the enhancement of human health, particularly for neurological and visual functions. Different modes of cultivation (batch, fed-batch and repeated-batch) by Aurantiochytrium sp. SW1 were studied for effective docosahexaenoic acid (DHA) production. In this study, three different modes of fermentation were carried out in 1 L shake flasks with a working volume of 500 mL, incubated at 30 ºC and 200 rpm. Batch cultivation significantly exceeds the rest of cultivation modes, achieving maximal lipid and DHA concentrations of 11.22 g/L and 5.87 g/L, respectively, and DHA productivity of 0.061 g/L/h. Lipid and DHA concentration of the repeated-batch process decreased through the cycles for all three different types of replacement ratio (80, 90 and 95%). The average decrease percentage of DHA concentration for cycle one and cycle two were 21.76 and 32.52%, respectively. However, the fatty acid composition of lipids obtained in the cycles remained consistent with 16:0 and DHA being the most abundant fatty acids indicating that this mode of fermentation is highly useable for industrial applications.

Direct Isomaltulose Synthesis From Beet Molasses by Immobilized Sucrose Isomerase

Isomaltulose is becoming a focus as a functional sweetener for sucrose substitutes; however, isomaltulose production using sucrose as the substrate is not economical. Low-cost feedstocks are needed for their production. In this study, beet molasses (BM) was introduced as the substrate to produce isomaltulose for the first time. Immobilized sucrose isomerase (SIase) was proved as the most efficient biocatalyst for isomaltulose synthesis from sulfuric acid (H2SO4) pretreated BM followed by centrifugation for the removal of insoluble matters and reducing viscosity. The effect of different factors on isomaltulose production is investigated. The isomaltulose still achieved a high concentration of 446.4 ± 5.5 g/L (purity of 85.8%) with a yield of 0.94 ± 0.02 g/g under the best conditions (800 g/L pretreated BM, 15 U immobilized SIase/g dosage, 40°C, pH of 5.5, and 10 h) in the eighth batch. Immobilized SIase used in repeated batch reaction showed good reusability to convert pretreated BM into isomaltulose since the sucrose conversion rate remained 97.5% in the same batch and even above 94% after 11 batches. Significant cost reduction of feedstock costs was also confirmed by economic analysis. The findings indicated that this two-step process to produce isomaltulose using low-cost BM and immobilized SIase is feasible. This process has the potential to be effective and promising for industrial production and application of isomaltulose as a functional sweetener for sucrose substitute.