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L-Malic acid (L-MA) is widely used in food and non-food products. However, few microorganisms have been able to efficiently produce L-MA from xylose derived from lignocellulosic biomass (LB). The objective of this work is to convert LB into L-MA with the concept of a bioeconomy and environmentally friendly process. The unique trifunctional xylanolytic enzyme, PcAxy43A from Paenibacillus curdlanolyticus B-6, effectively hydrolyzed xylan in untreated LB, especially corn hull to xylose, in one step. Furthermore, the newly isolated, Acetobacter tropicalis strain H1 was able to convert high concentrations of xylose derived from corn hull into L-MA as the main product, which can be easily purified. The strain H1 successfully produced a high L-MA titer of 77.09 g/l, with a yield of 0.77 g/g and a productivity of 0.64 g/l/h from the xylose derived from corn hull. The process presented in this research is an efficient, low-cost and environmentally friendly biological process for the green production of L-MA from LB.

The aim of this study was the experimental demonstration and assessment of a novel lignocellulose biorefinery (LCB) for the integration of beech wood-based products as platform and fine chemicals. The process sequence included organosolv pulping followed by pulp bleaching, hydrothermal conversion of hemicellulose to xylose and its purification, fermentation of xylose to malic acid, and base-catalyzed lignin depolymerization (BCD). The resulting products were dissolving pulp, phenolic BCD-oligomers, and malic acid. The state of the art for these technologies is their experimental proof of concept and validation at a laboratory- and pilot-scale and has a technology readiness level (TRL) of 3–4. By integrating and optimizing the single-process steps into one LCB, the TRL could be increased to 5. Based on the findings of the experimental studies, a LCB converting 50,000 dry metric tonnes ($$ \hat{=} $$ = ̂ 38.7 MW) of beech wood annually was simulated with Aspen Plus. Mass and energy balances showed that 14,616 dry metric tonnes of dissolving pulp, 5174 dry metric tonnes of BCD-oligomers, and 4077 dry metric tonnes of malic acid annually could be produced. The total energy efficiency is 40.3%. The calculation of specific production costs demonstrated the marketability of dissolving pulp (1350 €/t) and BCD-oligomers (2180 €/t), whereas malic acid (4750 €/t) is not yet competitive. Environmental assessment showed reduced greenhouse gas (GHG) emissions from the production of BCD-oligomers and malic acid and higher GHG emissions from the production of dissolving pulp compared with the reference products. In total, the examined LCB would contribute to the mitigation of global warming.

Background Consolidated bioprocessing (CBP) technique is a promising strategy for biorefinery construction, producing bulk chemicals directly from plant biomass without extra hydrolysis steps. Fixing and channeling CO2 into carbon metabolism for increased carbon efficiency in producing value-added compounds is another strategy for cost-effective bio-manufacturing. It has not been reported whether these two strategies can be combined in one microbial platform. Results In this study, using the cellulolytic thermophilic fungus Myceliophthora thermophila, we designed and constructed a novel biorefinery system DMCC (Direct microbial conversion of biomass with CO2 fixation) through incorporating two CO2 fixation modules, PYC module and Calvin–Benson–Bassham (CBB) pathway. Harboring the both modules, the average rate of fixing and channeling 13CO2 into malic acid in strain CP51 achieved 44.4, 90.7, and 80.7 mg/L/h, on xylose, glucose, and cellulose, respectively. The corresponding titers of malic acid were up to 42.1, 70.4, and 70.1 g/L, respectively, representing the increases of 40%, 10%, and 7%, respectively, compared to the parental strain possessing only PYC module. The DMCC system was further improved by enhancing the pentose uptake ability. Using raw plant biomass as the feedstock, yield of malic acid produced by the DMCC system was up to 0.53 g/g, with 13C content of 0.44 mol/mol malic acid, suggesting DMCC system can produce 1 t of malic acid from 1.89 t of biomass and fix 0.14 t CO2 accordingly. Conclusions This study designed and constructed a novel biorefinery system named DMCC, which can convert raw plant biomass and CO2 into organic acid efficiently, presenting a promising strategy for cost-effective production of value-added compounds in biorefinery. The DMCC system is one of great options for realization of carbon neutral economy.

In the present study, three north-western Himalayan medicinal plants, Ocimum sanctum, Terminalia bellerica and Terminalia chebula were used to evaluate the effectiveness of choline chloride based natural deep eutectic solvents (NADESs) in extracting the polyphenolic compounds. The natural deep eutectic solvents used in the study were choline chloride:glucose: water, choline chloride:fructose:water, choline chloride: xylose: water, choline chloride:glycerol:water and choline chloride:malic acid:water.The results of the above study showed that all the eutectic solvents were more effective in extracting phenolic compounds than methanol, however, choline chloride:malic acid:water based NADES was most effective among all the NADESs tested and methanol. Additionally, profiling of phenolic compounds in extracts obtained by choline chloride:malic acid:water and methanol was also carried out using HPLC analysis. It was also observed that extracts possessing higher polyphenolic content showed higher DPPH radical scavenging activity, ferric reducing antioxidant power, reducing power and α-glucosidase inhibitory activity. Our results suggests that natural deep eutectic solvents offers efficient and safe alternative to methanol for extraction of phenolic compounds from medicinal plants.

Camellia oleifera shell (COS), a by-product of processing woody vegetable oil, is rich in hemicellulose and lignin. In this study, we investigated the effects of acid concentration, pretreatment temperature and reaction time on the concentration and yield of xylo-oligosaccharides (XOS) and the degree of polymerization (DP) distribution of XOS when pretreating COS with malic acid (MA). Under moderate condition (2 M MA, 120 ℃, 30 min), the maximum yield of XOS with DP 2–4 was 48.78% (based on the initial xylan) with low xylose, 5-hydroxymethylfurfural (HMF) and furfural, in which xylobiose (X2), xylotriose (X3) and xylotraose (X4) concentrations were 5.22 g/L, 2.75 g/L and 2.91 g/L, respectively. In addition, acid-insoluble lignin (AIL) in the residue after MA pretreatment and milling wood lignin (MWL) were mainly composed of guaiacyl and syringyl. AIL has higher thermal stability than MWL, which can be the stabilizer for producing flame-resistant materials.

Polymalic acid (PMA) is a water-soluble biopolymer with many attractive properties for pharmaceutical applications. Its monomer, l -malic acid, is a potential C4 platform chemical, which is widely used in the food industry. In this study, a new strain with a high PMA yield from xylose was isolated and identified as Aureobasidium pullulans based on the morphological and phylogenetic analyzes. This strain produced PMA from xylose at a comparable or higher rate than glucose, and PMA produced from xylose had a similar molecular weight to that produced from glucose. A high final PMA titer of 80.4 g/L (91.2 g/L of malic acid after hydrolysis) was achieved using fed-batch fermentation with xylose in a 5-L fermentor at a PMA productivity of 0.52 g/L h. Corncob hydrolysate could be better utilized producing 28.6 g/L of PMA (or 32.4 g/L malic acid) in batch fermentation. PMA was further characterized for its chemical structure. This strain can be used to produce PMA and malic acid from xylose and lignocellulosic biomass hydrolysates.

The effects of the high-intensity ultrasound (HIUS) technology at the nominal powers of 300, 600, 900, and 1200 W were evaluated on the chemical stability of xylooligosaccharides (XOS) used to enrich orange juice. The ultrasound energy performance for each nominal power applied to the XOS-enriched orange juice was determined by calculating acoustic powers (W), HIUS intensity (W/cm2), and energy density (kJ/mL). Physicochemical properties (pH and soluble solid content), organic acid content (ascorbic, malic, and citric acids), total phenolic content (TPC), antioxidant activity by the FRAP (Ferric reducing ability of plasma) method, sugar (glucose, fructose, and sucrose), and XOS (xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose) content were determined. The pH and soluble solid content did not change after all HIUS treatments. The HIUS process severity was monitored by quantifying ascorbic acid content after the treatments. A significant linear decrease in the ascorbic acid content was observed in prebiotic orange juice with the HIUS process intensification by increasing nominal power. The malic acid and citric acid contents had similar behavior according to the HIUS process intensification. The nominal power increase from 300 to 600 W increased the concentration of both organic acids, however, the intensification up to 1200 W reduced their concentration in the functional beverage. The TPC and FRAP data corroborated with the results observed for the ascorbic acid content. However, the HIUS processing did not alter sugar and XOS contents. The XOS chromatographic profiles were not modified by the HIUS treatment and presented the same amount of all prebiotic compounds before and after the HIUS treatment. Overall, HIUS technology has been evaluated as a promising stabilization technique for prebiotic beverages enriched with XOS due to their high chemical stability to this emerging technology under severe process conditions.

In this study, 29 strains of Kluyveromyces marxianus with peculiar genetic and phenotypic traits previously isolated from a fermented goat milk of Yaghnob valley were investigated for chromosome length polymorphism (CLP) by PFGE, adhesion properties and carbon usage by Biolog analysis. Obtained data showed that strains differed in terms of number and size of chromosome bands. The number of bands ranged from 5 to 7, suggesting a probable genome size from 1.4 to 2.6 Mb. Strains showed a certain level of cell surface hydrophobicity ranging from 32% to 77.7%. Strains were also tested for their ability to form a biofilm on polystyrene plates: planktonic cells ranged from 6.3 cfu/mL to 7.95 cfu/mL, while sessile from 7.11 cfu/mL to 8.6 cfu/mL. The strains able to adhere to polystyrene plates were also able to form a mature MAT. Biolog analysis revealed that almost all strains were able to use putrescine, malic acid, α-D lactose, phenylethylamine, β-methyl D-gucoside and xylose; 5 strains were able to grow on cellobiose and 3 were able to catabolise α-ketobutyric. The obtained data highlighted a number of interesting features underlying the peculiar capacities of these strains for industrial applications.

Background C4-dicarboxylic acids, including malic acid, fumaric acid and succinic acid, are valuable organic acids that can be produced and secreted by a number of microorganisms. Previous studies on organic acid production by Aspergillus carbonarius, which is capable of producing high amounts of citric acid from varieties carbon sources, have revealed its potential as a fungal cell factory. Earlier attempts to reroute citric acid production into C4-dicarboxylic acids have been with limited success. Results In this study, a glucose oxidase deficient strain of A. carbonarius was used as the parental strain to overexpress a native C4-dicarboxylate transporter and the gene frd encoding fumarate reductase from Trypanosoma brucei individually and in combination. Impacts of the introduced genetic modifications on organic acid production were investigated in a defined medium and in a hydrolysate of wheat straw containing high concentrations of glucose and xylose. In the defined medium, overexpression of the C4-dicarboxylate transporter alone and in combination with the frd gene significantly increased the production of C4-dicarboxylic acids and reduced the accumulation of citric acid, whereas expression of the frd gene alone did not result in any significant change of organic acid production profile. In the wheat straw hydrolysate after 9 days of cultivation, similar results were obtained as in the defined medium. High amounts of malic acid and succinic acid were produced by the same strains. Conclusions This study demonstrates that the key to change the citric acid production into production of C4-dicarboxylic acids in A. carbonarius is the C4-dicarboxylate transporter. Furthermore it shows that the C4-dicarboxylic acid production by A. carbonarius can be further increased via metabolic engineering and also shows the potential of A. carbonarius to utilize lignocellulosic biomass as substrates for C4-dicarboxylic acid production.

A purple non-sulfur (PNS) photosynthetic bacterium was isolated from an upflow anaerobic sludge blanket (UASB) bioreactor for methane production and was identified as Rhodobacter sphaeroides KKU-PS5 (GenBank Accession no. KC481702) by 16s rRNA gene sequence analysis. Strain KKU-PS5 could utilize glucose, xylose, fructose, arabinose, malate, succinate, acetate, butyrate, lactate and D-mannitol for growth and hydrogen production. Malate was a preferred carbon source while glutamate and Aji-L (waste from the process of crystallizing monosodium glutamate) were the preferred nitrogen sources. The ability to utilize Aji-L as a low-cost nitrogen supplement for photo-biohydrogen production by the strain KKU-PS5 is considered as its desirable characteristic. The threshold substrate concentration of malate was 30 mmol/L. The optimum conditions for hydrogen production from malate were an initial pH of 7.0, FeSO4 concentration of 4 mg/L, temperature of 30 °C and light intensity of 6 klux. Under the optimum conditions, the maximum hydrogen production, the hydrogen yield (HY) and the hydrogen production rate (HPR) of 1330 mL-H2/L, 3.80 mol-H2/mol-malate, and 11.08 mL-H2/L h, respectively, were achieved. Hydrogen production under a dark/light cycle led to a decreased HY and HPR in comparison to continuous illumination.

The microbial fermentation of malic acid, which is one of the most important organic acid platforms used widely in food and chemical engineering, has attracted considerable interest. A malate production strain was isolated, a mutation was induced, and regulation of the metabolic network was then conducted. The identification results showed that the malic acid production strain, HF- 119, belonged to Rhizopus delemar. An analysis of the metabolic pathway showed that the malic acid flux of this strain occurred through three main pathways, and many byproducts, such as succinic acid, fumaric acid and ethanol, were produced. Although corn straw hydrolyte was used, the metabolism of xylose was not as rapid as that of glucose. Subsequently, breeding of the strains and regulation of the metabolic network resulted in an increase in malate yield, and the strain HF-121 produced more than 120 g/L malic acid within 60 h. The ability to produce malic acid from biomass hydrolyte highlights the industrial development potential of this strain.

A new species of the genus Ancylobacter, exemplified by strain Z-0056, was isolated from dystrophic humified waters formed by xylotrophic fungi grown on decaying spruce wood. The cells of strain Z-0056 (0.65–0.9 μm) are coccoid, gram-negative, with fimbriae, and nonmotile. The strain is pleomorphic and reproduces by nonuniform division. Strain Z-0056 is an aerobic organoheterotroph and a mesophilic and moderately acidophilic oligotrophic microorganism. As an inhabitant of dystrophic ultrafresh waters, strain Z-0056 is sensitive to NaCl. The bacterium utilizes organic acids (acetate, pyruvate, oxalate, gluconate, malate, succinate, and citrate), as well as xylose and xylan. The microorganism grows in a pH range of 4.0–8.0, with an optimum at pH 5.5. The temperature range for growth is 15–25°C, with an optimum at 20°C. According to its ecophysiological properties, strain Z-0056 belongs to the group of ombrophilic dissipotrophs. The DNA G+C base content is 66.8 mol %. According to phylogenetic analysis, strain Z-0056 belongs to the genus Ancylobacter. Strain Z-0056 showed the highest similarity (98.3%) with the type strain of the species A. oerskovii. The phenotypic and phylogenetic properties of strain Z-0056 support classification of this microorganism within the genus Ancylobacter as the novel species Ancylobacter abiegnus sp. nov.

Malic acid is a C4 dicarboxylic acid that is currently mainly used in the food and beverages industry as an acidulant. Because of the versatility of the group of C4 dicarboxylic acids, the chemical industry has a growing interest in this chemical compound. As malic acid will be considered as a bulk chemical, microbial production requires organisms that sustain high rates, yields, and titers. Aspergillus oryzae is mainly known as an industrial enzyme producer, but it was also shown that it has a very competitive natural production capacity for malic acid. Recently, an engineered A. oryzae strain, 2103a-68, was presented which overexpressed pyruvate carboxylase, malate dehydrogenase, and a malic acid transporter. In this work, we report a detailed characterization of this strain including detailed rates and yields under malic acid production conditions. Furthermore, transcript levels of the genes of interest and corresponding enzyme activities were measured. On glucose as carbon source, 2103a-68 was able to secrete malic acid at a maximum specific production rate during stationary phase of 1.87 mmol (g dry weight (DW))−1 h−1 and with a yield of 1.49 mol mol−1. Intracellular fluxes were obtained using 13C flux analysis during exponential growth, supporting the success of the metabolic engineering strategy of increasing flux through the reductive cytosolic tricarboxylic acid (rTCA) branch. Additional cultivations using xylose and a glucose/xylose mixture demonstrated that A. oryzae is able to efficiently metabolize pentoses and hexoses to produce malic acid at high titers, rates, and yields.

l-Malic acid and fumaric acid are C4 dicarboxylic organic acids and considered as promising chemical building blocks. They can be applied as food preservatives and acidulants in rust removal and as polymerization starter units. Molds of the genus Aspergillus are able to produce malic acid in large quantities from glucose and other carbon sources. In order to enhance the production potential of Aspergillus oryzae DSM 1863, production and consumption rates in an established bioreactor batch-process based on glucose were determined. At 35 °C, up to 42 g/L malic acid was produced in a 168-h batch process with fumaric acid as a by-product. In prolonged shaking flask experiments (353 h), the suitability of the alternative carbon sources xylose and glycerol at a carbon-to-nitrogen (C/N) ratio of 200:1 and the influence of different C/N ratios in glucose cultivations were tested. When using glucose, 58.2 g/L malic acid and 4.2 g/L fumaric acid were produced. When applying xylose or glycerol, both organic acids are produced but the formation of malic acid decreased to 45.4 and 39.4 g/L, respectively. Whereas the fumaric acid concentration was not significantly altered when cultivating with xylose (4.5 g/L), it is clearly enhanced by using glycerol (9.3 g/L). When using glucose as a carbon source, an increase or decrease of the C/N ratio did not influence malic acid production but had an enormous influence on fumaric acid production. The highest fumaric acid concentrations were determined at the highest C/N ratio (300:1, 8.44 g/L) and lowest at the lowest C/N ratio (100:1, 0.7 g/L).

The organic acid producer Aspergillus oryzae and Rhizopus delemar are able to convert several alternative carbon sources to malic and fumaric acid. Thus, carbohydrate hydrolysates from lignocellulose separation are likely suitable as substrate for organic acid production with these fungi. Before lignocellulose hydrolysate fractions were tested as substrates, experiments with several mono- and disaccharides, possibly present in pretreated biomass, were conducted for their suitability for malic acid production with A. oryzae. This includes levoglucosan, glucose, galactose, mannose, arabinose, xylose, ribose, and cellobiose as well as cheap and easy available sugars, e.g., fructose and maltose. A. oryzae is able to convert every sugar investigated to malate, albeit with different yields. Based on the promising results from the pure sugar conversion experiments, fractions of the organosolv process from beechwood (Fagus sylvatica) and Miscanthus giganteus were further analyzed as carbon source for cultivation and fermentation with A. oryzae for malic acid and R. delemar for fumaric acid production. The highest malic acid concentration of 37.9 ± 2.6 g/L could be reached using beechwood cellulose fraction as carbon source in bioreactor fermentation with A. oryzae and 16.2 ± 0.2 g/L fumaric acid with R. delemar. We showed in this study that the range of convertible sugars for A. oryzae is even higher than known before. We approved the suitability of fiber/cellulose hydrolysate obtained from the organosolv process as carbon source for A. oryzae in shake flasks as well as in a small-scale bioreactor. The more challenging hemicellulose fraction of F. sylvatica was also positively evaluated for malic acid production with A. oryzae.