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Deep eutectic solvents (DES) have been employed for the pretreatment of grape pomace and stalks within a biorefinery concept. Four DES, produced with choline chloride (ChCl) as hydrogen bond acceptor and four carboxylic acids as hydrogen bond donors, namely formic acid (FA), acetic acid (AA), lactic acid (LA) and oxalic acid (OA), were evaluated considering lignin removal efficiency, polysaccharide hydrolysis efficiency into C5 and C6 sugars, recyclability and reusability. The mixture of ChCl:LA at 1:10 molar ratio, 120°C and 1 h pretreatment, led to 40% lignin removal over four pretreatment cycles. Enzymatic hydrolysis of the remaining solids after the first pretreatment cycle resulted in 92.7% glucan and 36.6% hemicellulose hydrolysis yield. The hydrolysate was used as fermentation feedstock in batch Actinobacillus succinogenes bioreactor cultures leading to 36 g/L succinic acid with a yield of 0.62 gSA per g total sugars and 0.65 g/(L⋅h) productivity. Using 2 kg of grape pomace and stalks in each pretreatment cycle, the succinic acid that could be produced in five consecutive cycles is 200.8 g, 208 g, 204.9 g, 184.5 g and 94.3 g. Graphical Abstract

Research on grape microbiome dynamics inaugurates new perspectives for sophisticated complex bio-transformations that can profoundly enhance wine character. The exploitation of the significant variations in yeast biota of grapevines in different viticultural areas entails the potential to confer specific regional character in wines. Thereby, numerous studies are following this flourishing oenological interest on distinct microbial fingerprints and dynamic interactions over fermentation. Premium quality ‘natural’ wines are an emerging topic of the current decade in winemaking. Thus, research is directed towards the characterization of genotypes of indigenous yeasts and their beneficial role in unique wine profiles. The identification of new fermentative yeast strains undertakes continuous challenges in the production of top-of-the-line wines with exclusive regional traits. Herein, distinctive major trends in winemaking are reviewed whereas novel and promising studies are being highlighted.

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.

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.

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 g

The fluctuation in the price of crude oil during the last decades, the importance of resource sustainability, and the environmental concerns related to plastics pollution have promoted the research and development of bio-based plastics. Among them, polyhydroxyalkanoates (PHA) have been attracting interest for the past decades as they are 100% bio-based, biocompatible, and biodegradable polymers that closely resemble the properties of conventional polymers such as poly(propylene) (PP) or low-density poly(ethylene) (LDPE). Over the past several years, their sustainable production in a biorefinery context, tailored microstructure and properties, and wide range of applications have been extensively researched. In this study, the advantages of PHA against other bio-based and conventional polymers are discussed; current applications of PHA-based polymers are presented, highlighting innovative products already available on the market, and the potential use of PHA monomers is addressed.

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.

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.

The bacterial strain Enterobacter ludwigii FMCC 204 was selected as the most efficient 2,3-butanediol (BDO) producer among five strains when cultivated on glucose, fructose, mannose, arabinose, xylose, galactose and sucrose in shake flask cultures. Plums, apples and pears were used for the production of fermentation medium via sequential maceration, suspension in water and centrifugation to collect the supernatant. Sugar production from mixed vegetables was evaluated via sulphuric acid treatment leading to 65.8% (w/w) of hemicellulose hydrolysis yield at initial solid concentrations of 50 g/L (on a dry basis) treated with 3% (v/v) H2SO4. Fed-batch cultures of E. ludwidgii on fruit derived feedstock resulted in BDO concentration, yield and productivity of 50 g/L, 0.4 g/g and 0.41 g/L/h. BDO production from vegetable waste hydrolysates via fed-batch cultures led to BDO concentration of 17.6 g/L. This study demonstrated that fruit and vegetable wastes from open markets can be used as fermentation feedstocks for BDO production.

A novel bio-economy business model for production of bio-based succinic acid and value-added co-products has been developed using biodiesel industry by-products. Sunflower meal (SFM) fractionation resulted in an antioxidant-rich extract via aqueous ethanol extraction and a protein concentrate stream via NaOH recycling using ultrafiltration. Solid state fermentation (SSF) by Aspergillus awamori using untreated SFM resulted in the production of cellulase, β-glucosidase, xylanase, protease and phytase. After the extraction of co-products, the residual SFM was hydrolysed via thermochemical and enzymatic treatment using crude enzymes resulting in conversion yields of 0.23 g g−1 total Kjeldahl nitrogen to free amino nitrogen, 0.56 g g−1 cellulose to glucose and 0.92 g g−1 hemicellulose to sugars. The SFM-derived hydrolysate was used in fed-batch cultures of Actinobacillus succinogenes leading to 34 g L−1 succinic acid with a yield of 0.6 g g−1 and a productivity of 0.71 g L−1 h−1. Crude glycerol supplemented with SFM hydrolysate resulted in 69.1 g L−1 succinic acid with a yield of 0.39 g g−1 and a productivity of 1.26 g L−1 h−1 by Yarrowia lipolytica PSA02004. SFM was efficiently used for co-product extraction, fermentation medium production and on-site crude enzyme production via SSF.

The development of cost-competitive bioprocesses is dependent on the formulation of low cost and efficient fermentation medium. Thus, optimization of nutrient composition in fermentation media combining efficient metabolic product formation with cost-competitiveness is needed. Within this context, the main novelty of this study was to optimize the nutrient composition of a fermentation medium used for bio-based succinic acid production by the wild-type bacterial strain Basfia succiniciproducens JF4016 combining low cost with high succinic acid production efficiency. Two Plackett-Burman Designs, a central composite design and constraint optimization were sequentially implemented to estimate the optimum and cost-competitive fermentation medium composition. The two Plackett-Burman Designs identified NaH 2PO4, Na2HPO4 and NaCl as the most important medium components. Central composite design identified the optimum concentrations of NaH 2PO4, Na2HPO4, NaCl, yeast extract and corn steep liquor leading to the highest productivity. Constraint optimization was employed to maximise productivity using constraints on the final succinic acid concentration, yield, by-products to succinic acid ratio and fermentation medium cost Validation of the model predictions was carried out in a bioreactor culture at ca. 30 g/L initial glucose concentration. The optimum fermentation medium containing 19.4 g/L CSL, 9.2 g/L yeast extract, 3 g/L NaCl and 4.32 g/L sodium phosphates led to the production of 19 g/L succinic acid concentration, 0.62 g/g yield and 1.9 g/(L ⋅ h) productivity.

The utilization of crude waste streams and the development of novel bioprocessing routes are necessary in order to expand the industrial implementation of bio-based succinic acid production. This work focuses on the utilization of zero cost municipal organic biowaste for the production of bio-based succinic acid using the engineered yeast strain Yarrowia lipolytica PSA02004. Fermentation advances focused on pH regulation towards reduced NaOH requirements. Optimum initial carbon source concentration (100–120 g/L) in crude hydrolysates was estimated in shake flask cultures. Optimum kLα (183.2 h−1) in fed-batch cultures resulted in 42.2 g/L succinic acid with 0.38 g/g yield and 0.84 g/L/h productivity. A two-stage pH regulation strategy was employed to enhance succinic acid production efficiency and reduce NaOH requirements. The gradual reduction of pH from 6 to 5.5 resulted in 54.4 g/L succinic acid with 0.44 g/g yield and 0.82 g/L/h productivity and 43% lower NaOH consumption. The minimum selling price of succinic acid was estimated at $2.7/kg and $2.94/kg at annual production capacity of 50,000 t when the production cost of organic biowaste derived sugars were considered at $10/tTS and $110/tTS, respectively. The global warming potential and abiotic depletion potential were estimated at 2.72 kgCO2-eq/kgSA and 33.4 MJ/kgSA, respectively. Results showed that pH regulation is an important operating parameter towards the sustainable succinic acid production using municipal organic biowaste.

The fermentative production of succinic acid leads to increasing toxicity over time, and requires base addition to counteract acidification. Here we integrated a fed-batch Basfia succiniciproducens succinic acid fermentation with membrane electrolysis. This approach brings the broth in direct contact with an OH- and H-2 producing cathode, and enables in situ extraction of succinate towards the low volume H+ and O-2 producing anode compartment. In the latter, the succinate is acidified to succinic acid and precipitated. The key advantage of the cathodic process is the production of base maintaining fermentation pH with limited external base addition. The bacterial cells are recycled through the cathode compartment of the electrochemical cell, and exposed to H-2, creating biological reducing power. Fermentations were executed with glucose, xylose and ultrafiltered spent sulphite liquor (SSL), the side stream generated via acidic sulphite pulping of Eucalyptus globulus wood. The membrane was not permeable to cells, color and solids, which resulted in combined succinate extraction, clarification, acidification and concentration in a single unit operation. The succinic acid to by-product ratio increased, favoring the production of succinic acid over lactic, formic and acetic acids, with up to 15% higher total sugars to succinic acid conversion yield. The OH-production due to water reduction resulted in lower NaOH usage (up to 33% less) for maintaining the pH during fermentation. The maximum productivity also increased by 30% to a rate of 0.41 g L-1 h(-1) with the electrochemical system, with 1.65 kW h per kg succinic acid extracted in the case of SSL and 2.4 and 1.9 kW h per kg succinic acid extracted in the case of glucose and xylose, respectively. Considering the aforementioned advantages, this integrated system could evolve into a breakthrough technology in sustainable industrial succinic acid production from crude industrial side streams.

This work focuses on using renewable sources as feedstock for the microbial production of carotenoids, valuable compounds for various industries. Xanthophyllomyces dendrorhous, which mainly synthesize astaxanthin and β-carotene, was used for the process. We proposed two strategies to achieve high production yields, the correct oxygen supply to the medium and the adoption of a fed-batch mode. Two factors related to the medium's oxygen transfer at a flask scale (working volume and agitation) were evaluated. Astaxanthin production was successfully correlated with the oxygen transfer coefficient (kLa) using a mesquite pods-based medium (MEM) and a chemically defined one (YM). kLa correlations were then used to set the conditions at a reactor scale. An optimum kLa of 16.86 h−1 was adequate to promote astaxanthin production in MEM (1824.13 μg/L). Meanwhile, in YM, optimum production (1656.24 μg/L) was obtained at the highest evaluated levels (17.77 h−1). Thus, a higher oxygen transfer rate is required to produce carotenoids in YM compared with MEM. Astaxanthin production was thus evaluated using a fed-batch process at flask and reactor scales. Reached production levels with fed-batch cultivation were 4465.12 μg/L (flask) and 4130.23 μg/L (reactor). This study demonstrated the correlation between oxygen transfer and carotenoids' production and the potential utilization of mesquite feedstock in a fed-batch bioprocess to improve carotenoids' yield by X. dendrorhous.

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.

This study focuses on the development of unstructured models, including both substrate and product inhibition, that predict the cultivation of Actinobacillus succinogenes and Basfia succiniciproducens on a mixture of C5 and C6 sugars, similar to the sugar composition contained in spent sulphite liquor, the liquid waste stream from the sulphite pulping process. The main sugar monomer contained in the medium was xylose (72.6%) with galactose (12.2%), glucose (10.9%), mannose (4.2%) and arabinose (0.1%) making up the remaining sugar content. The growth inhibition caused by metabolic products (succinic, lactic, acetic, formic and mixed acids) and initial mixed sugar concentration was determined. The highest obtained succinic acid yield, final concentration and productivity in fermentations carried out in Duran bottles were 0.76 g/g, 26.0 g/L and 0.66 g/L/h for B. succiniciproducens and 0.69 g/g, 27.4 g/L and 0.60 g/L/h for A. succinogenes, respectively (the units in yield calculations are referred to grams of succinic acid produced per gram of total sugars consumed). The kinetic parameters for both strains were estimated from experimental results. The obtained R2 values for the fitted models were 0.96 for A. succinogenes and 0.94 for B. succiniciproducens. A sensitivity analysis on the obtained parameters showed that the maximum specific growth rates (μmax) and the growth associated substrate consumption parameters (γ) are the most influential model parameters for both microorganisms. The model was validated by fermentations conducted in lab-scale bioreactors showing good agreement between experimental data and model simulations.

Actinobacillus succinogenes is a wild-type bacterial strain, isolated from bovine rumen, known as one of the most efficient natural producers of succinic acid. Herein, the factors contributing to the fermentative production of succinic acid by A. succinogenes are reviewed with particular focus on raw materials, culture conditions, significance of carbon dioxide availability and downstream separation and purification. The metabolic potential of this strain is evaluated through discussion of the pathways involved in succinic acid production, genome analysis as well as the development of A. succinogenes mutants. The review also addresses the importance of by-product formation during fermentation that constitutes an important aspect regulating succinic acid production by A. succinogenes. The prospect of integrating succinic acid production in future biorefineries is assessed.

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.

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.