Transcriptional regulation in key metabolic pathways of Actinobacillus succinogenes in the presence of electricity.

The potential of renewable energy application via direct electrode interaction for the production of bio-based chemicals is a promising technology. The utilization of extracellular energy in pure culture fermentations aims in intracellular redox balance regulation in order to improve fermentation efficiency. This work evaluates the impact of a bioelectrochemical system in succinic acid fermentation and the metabolic response of Actinobacillus succinogenes. The metabolic pathway regulation of A. succinogenes was evaluated via RNA expression of the key enzymes that participate in TCA cycle, pyruvate metabolism and oxidative phosphorylation. The genes that were significantly overexpressed in BES compared to non-BES were phosphoenolpyruvate carboxykinase (0.4-fold change), inorganic pyrophosphatase (2.3-fold change) and hydrogenase (2.2-fold change) and the genes that were significantly underexpressed were fumarase (-0.94-fold change), pyruvate kinase (-6.9-fold change), all subunits of fumarate reductase (-2.1 to -1.17-fold change), cytochromes I and II (-1.25 and -1.02-fold change, respectively) and two C4-carboxylic acid transporters.

Fermentative bioconversion of food waste into biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) using Cupriavidus necator.

Dependency on plastic commodities has led to a recurrent increase in their global production every year. Conventionally, plastic products are derived from fossil fuels, leading to severe environmental concerns. The recent coronavirus disease 2019 pandemic has triggered an increase in medical waste. Conversely, it has disrupted the supply chain of personal protective equipment (PPE). Valorisation of food waste was performed to cultivate C. necator for fermentative production of biopolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The increase in biomass, PHBV yield and molar 3-hydroxy valerate (3HV) content was estimated after feeding volatile fatty acids. The fed-batch fermentation strategy reported in this study produced 15.65 ± 0.14 g/L of biomass with 5.32 g/L of PHBV with 50% molar 3HV content. This is a crucial finding, as molar concentration of 3HV can be modulated to suit the specification of biopolymer (film or fabric). The strategy applied in this study addresses the issue of global food waste burden and subsequently generates biopolymer PHBV, turning waste to wealth.

Biorefinery development, techno-economic evaluation and environmental impact analysis for the conversion of the organic fraction of municipal solid waste into succinic acid and value-added fractions.

The organic fraction of municipal solid waste (OFMSW) was used for biorefinery development within a circular bioeconomy context towards extraction of lipids/fats and proteins with 100% and 68% recovery yields, respectively, as well as succinic acid (SA) production. A nutrient-rich hydrolysate (89.1 g/L sugars) produced using crude enzymes derived via solid state fermentation of Aspergillus awamori, was employed in Actinobacillus succinogenes fermentation leading to 31.7 gSA/L with 0.68 g/g yield and 0.67 g/L/h productivity. The SA minimum selling price ($1.13-2.39/kgSA) considering 60,000 tSA/year production varied depending on co-product market prices and OFMSW management fees. The biorefinery using 1000 kg OFMSW contributes 35% lower CO2 emissions than conventional processes for the production of 105 kg vegetable oil, 87 kg vegetable protein and 206.4 kg fossil-SA considering also the CO2 emissions due to OFMSW landfilling. The proposed OFMSW biorefinery leads to cost-competitive SA production with lower CO2 emissions for OFMSW treatment.

Integrated biorefinery development using winery waste streams for the production of bacterial cellulose, succinic acid and value-added fractions.

An integrated biorefinery has been developed using winery wastes (grape pomace-GP, stalks-GS, wine lees-WL). Bacterial cellulose was produced from GP extracted free sugars. Grape-seed oil and polyphenols were extracted from GP. Experimental design was employed to optimize lignin removal (50.8%) from mixtures of remaining GP solids and GS via NaOH (1.19% w/v) treatment at 70°C for 30 min. Delignification liquid contained condensed tannins with 76% Stiasny number. Enzymatic hydrolysis produced a sugar-rich hydrolysate (40.2 g/L sugars). Ethanol, antioxidants, tartaric acid and nutrient-rich hydrolysate were produced from WL. The crude hydrolysates were used in fed-batch Actinobacillus succinogenes cultures for 37.2 g/L succinic acid production. The biorefinery produces 42.65 g bacterial cellulose, 24.3 g oil, 40.3 g phenolic-rich extract with 1.41 Antioxidant Activity Index, 80.2 g ethanol, 624.8 g crude tannin extract, 20.03 g tartaric acid and 157.8 g succinic acid from 1 kg of each waste stream.

Chemical Profiling, Bioactivity Evaluation and the Discovery of a Novel Biopigment Produced by Penicillium purpurogenum CBS 113139.

Biobased pigments are environmentally friendly alternatives to synthetic variants with an increased market demand. Production of pigments via fermentation is a promising process, yet optimization of the production yield and rate is crucial. Herein, we evaluated the potential of Penicillium purpurogenum to produce biobased pigments. Optimum sugar concentration was 30 g/L and optimum C:N ratio was 36:1 resulting in the production of 4.1-4.5 AU (namely Pigment Complex A). Supplementation with ammonium nitrate resulted in the production of 4.1-4.9 AU (namely Pigment Complex B). Pigments showed excellent pH stability. The major biopigments in Pigment Complex A were N-threonyl-rubropunctamin or the acid form of PP-R (red pigment), N-GABA-PP-V (violet pigment), PP-O (orange pigment) and monascorubrin. In Pigment Complex B, a novel biopigment annotated as N-GLA-PP-V was identified. Its basic structure contains a polyketide azaphilone with the same carboxyl-monascorubramine base structure as PP-V (violet pigment) and γ-carboxyglutamic acid (GLA). The pigments were not cytotoxic up to 250 µg/mL.

Succinic acid production from pulp and paper industry waste: A transcriptomic approach.

The fermentative production of biobased chemicals and polymers using crude lignocellulose hydrolysates is challenging due to the presence of various inhibitory compounds and multiple sugars. This study evaluates the metabolic response of Actinobacillus succinogenes for the production of succinic acid using spent sulphite liquor (SSL) as feedstock derived from industrial acidic sulphite pulping of Eucalyptus globulus hardwood. A transcriptomic approach led to significant insights on gene regulation of the major metabolic pathways (glycolysis, pentose phosphate pathway, TCA cycle, pyruvate metabolism and oxidative phosphorylation) in batch cultures carried out on SSL and compared with glucose and xylose. Significantly overexpressed genes in SSL compared to glucose and xylose were fructose biphosphate aldolase (> 1.18-fold change) in the catabolism, phosphoenolpyruvate carboxykinase (> 1.59-fold change) and malate dehydrogenase (> 1.49-fold change) in the TCA cycle, citrate lyase (> 1.7-fold change), dihydrolipoamide dehydrogenase (> 0.88-fold change), pyruvate dehydrogenase E2 (> 1.63-fold change) and pyruvate formate lyase (> 0.61-fold change), involved in acetyl-CoA pathways. Finally, C4 tricarboxylic transporters were overexpressed (DCU (> 1.61-fold change) and 0079 (> 4.19-fold change). SSL was responsible for the upregulation of genes involved in the TCA cycle and oxidative phosphorylation, while xylose showed similar results with SSL in the oxidative phosphorylation.

Sustainable production of bio-based chemicals and polymers via integrated biomass refining and bioprocessing in a circular bioeconomy context.

The sustainable production of bio-based chemicals and polymers is highly dependent on the development of viable biorefinery concepts using crude renewable resources for the production of diversified products. Within this concept, this critical review presents the availability of fractionated co-products and fermentable sugars that could be derived from major industrial and food supply chain side streams in EU countries. Fermentable sugars could be used for the production of bio-based chemicals and polymers. The implementation of biorefinery concepts in industry should depend on the evaluation of process efficiency and sustainability including techno-economic, environmental and social impact assessment following circular bioeconomy principles. Relevant sustainability indicators and End-of-Life scenarios have been presented. A case study on the techno-economic evaluation of bio-based succinic acid production from the organic fraction of municipal solid waste has been presented focusing on the evaluation of process profitability and feedstock requirements.

Evaluation of organic fractions of municipal solid waste as renewable feedstock for succinic acid production.

BACKGROUND: Despite its high market potential, bio-based succinic acid production experienced recently a declining trend because the initial investments did not meet the expectations for rapid market growth. Thus, reducing the succinic acid production cost is imperative to ensure industrial implementation. RESULTS: Succinic acid production has been evaluated using hydrolysates from the organic fraction of municipal solid waste (OFMSW) collected from MSW treatment plants. A tailor-made enzymatic cocktail was used for OFMSW hydrolysate production containing up to 107.3 g/L carbon sources and up to 638.7 mg/L free amino nitrogen. The bacterial strains Actinobacillus succinogenes and Basfia succiniciproducens were evaluated for succinic acid production with the latter strain being less efficient due to high lactic acid production. Batch A. succinogenes cultures supplemented with 5 g/L yeast extract and 5 g/L MgCO3 reached 29.4 g/L succinic acid with productivity of 0.89 g/L/h and yield of 0.56 g/g. Continuous cultures at dilution rate of 0.06 h-1 reached 21.2 g/L succinic acid with yield of 0.47 g/g and productivity of 1.27 g/L/h. Downstream separation and purification of succinic acid was achieved by centrifugation, treatment with activated carbon, acidification with cation exchange resins, evaporation and drying, reaching more than 99% purity. Preliminary techno-economic evaluation has been employed to evaluate the profitability potential of bio-based succinic acid production. CONCLUSIONS: The use of OFMSW hydrolysate in continuous cultures could lead to a minimum selling price of 2.5 $/kg at annual production capacity of 40,000 t succinic acid and OFMSW hydrolysate production cost of 25 $/t sugars.

Microbial oil production from various carbon sources by newly isolated oleaginous yeasts.

Microbial oil production has received significant attention as a potential precursor for the production of biofuels, oleochemicals and food products. In this study, six oleaginous yeasts, isolated from fruits, were selected based on their ability to accumulate high intracellular content of microbial oil (20-48% w/w of total dry weight). The highest content of saturated fatty acids was 68.7% (w/w), whereas the highest content of oleic acid was 62.7% (w/w). Furthermore, nutrient-rich hydrolysates produced via enzymatic hydrolysis of flour-rich waste streams generated by a confectionery industry were evaluated as fermentation media for microbial oil production via fed-batch bioreactor cultures using one of the most promising isolates, namely VV_D4. A total dry weight of 40 g/L with a microbial oil content of 39% (w/w) was produced by isolate VV_D4. Critical biodiesel properties were estimated based on the fatty acid composition and correlated with the international standards. The microbial oil produced by the new isolates could be potentially used for biodiesel production.

Pretreatment of spent sulphite liquor via ultrafiltration and nanofiltration for bio-based succinic acid production.

Ultrafiltration and nanofiltration of spent sulphite liquor (SSL) has been employed to evaluate the simultaneous production of lignosulphonates and bio-based succinic acid using the bacterial strains Actinobacillus succinogenes and Basfia succiniciproducens. Ultrafiltration with membranes of 10, 5 and 3kDa molecular weight cut-off results in significant losses of lignosulphonates (26-50%) in the permeate stream, while nanofiltration using membrane with 500Da molecular weight cut-off results in high retention yields of lignosulphonates (95.6%) in the retentate stream. Fed-batch bioreactor cultures using permeates from ultrafiltrated SSL resulted in similar succinic acid concentration (27.5g/L) and productivity (0.4g/L/h) by both strains. When permeates from nanofiltrated SSL were used, the strain B. succiniciproducens showed the highest succinic acid concentration (33.8g/L), yield (0.58g per g of consumed sugars) and productivity (0.48g/L/h). The nanofiltration of 1t of thick spent sulphite liquor could lead to the production of 306.3kg of lignosulphonates and 52.7kg of succinic acid, whereas the ultrafiltration of 1t of thick spent sulphite liquor using a 3kDa membrane could result in the production of 237kg of lignosulphonates and 71.8kg of succinic acid when B. succiniproducens is used in both cases.

Succinic acid production by Actinobacillus succinogenes from batch fermentation of mixed sugars.

Succinic acid production from the monosaccharides xylose, arabinose, glucose, mannose and galactose was studied using the bacterium Actinobacillus succinogenes. In Duran bottle cultures, containing 10 g/L of each of sugar, succinic acid was produced from all sugars except for galactose. The highest succinate yield, 0.56 g/g, was obtained with glucose, whereas the succinate yield was 0.42, 0.38 and 0.44 g/g for xylose, mannose and arabinose, respectively. The specific succinate productivity was 0.7 g/g h for glucose, but below 0.2 g/g h for the other sugars. Batch bioreactor fermentations were carried out using a sugar mixture of the five sugars giving a total concentration of 50 g/L, mimicking the distribution of sugars in spent sulfite liquor (SSL) from Eucalyptus which is rich in xylose. In this mixture, an almost complete conversion of all sugars (except galactose) was achieved resulting in a final succinate concentration of 21.8-26.8 g/L and a total yield of 0.59-0.68 g/g. There was evidence of co-consumption of glucose and xylose, whereas mannose was consumed after glucose. The main by-products were acetate 0.14-0.20 g/g and formate 0.08-0.13 g/g. NADH balance calculations suggested that NADH required for succinate production was not met solely from formate and acetate production, but other means of NADH production was necessary. Results from mixed sugar fermentations were verified using SSL as substrate resulting in a succinate yield of 0.60 g/g. In addition, it was found that CO2 sparging could replace carbonate supply in the form of MgCO3 without affecting the succinate yield.