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

The utilization of renewable resources for the production of bio-based products is essential in order to develop sustainable bioprocesses and biorefineries. Xylose-rich hydrolysates produced from hemicelluloses contained in lignocellulosic resources could be used for the production of succinic acid, one of the most important platform chemicals in the bio-economy era. Exploitation of spent sulphite liquor (SSL), a xylose rich by-product from pulp and paper industry has been previously investigated for succinic acid production by Actinobacillus succinogenes, one of the most efficient natural succinic acid producers. In this study, the metabolic potential of this strain was evaluated through the RNA expression of the metabolic pathways involved in succinic acid production utilizing glucose, xylose or SSL as carbon sources. A transcriptomic approach of the key enzymes of glucose and xylose catabolism, carboxylic acid production as well as oxidative phosphorylation led to an improved understanding on the energy consuming metabolic pathways. The transcriptomic analysis was carried out in batch cultures. A cDNA library was constructed at different phases of the fermentation where major metabolic changes in extracellular metabolites or biomass production were observed. Real time PCR was used to determine the expression levels of the genes of interest throughout the fermentation. The bottlenecks of the fermentative production of succinic acid by A. succinogenes were addressed with particular focus on the effect of glucose and xylose catabolism on pathways that involve ATP consumption and NADH oxidation. All subunits of ATP synthase were highly expressed in all substrates. In particular ATP synthase F0 (ATP SYN F0) was higher expressed when glucose was the only carbon source. Phosphoenol-pyruvate carboxykinase (PEPCK) expression was delayed when xylose was present in the medium. Despite the fact that extracellular lactic acid was not detected, low expression levels of lactic acid dehydrogenase (

Lipid production by yeasts growing on commercial xylose in submerged cultures with process water being partially replaced by olive mill wastewaters

Six yeast strains belonging to Rhodosporidium toruloides, Lipomyces starkeyi, Rhodotorula glutinis and Cryptococcus curvatus were shake-flask cultured on xylose (initial sugar – S0 =70±10 g/L) under nitrogen-limited conditions. C. curvatus ATCC 20509 and L. starkeyi DSM 70296 were further cultured in media where process waters were partially replaced by the phenol-containing olive-mill wastewaters (OMWs). In flasks with S0≈100 g/L and OMWs added yielding to initial phenolic compounds concentration (PCC0) between 0.0 g/L (blank experiment) and 2.0 g/L, C. curvatus presented maximum total dry cell weight - TDCWmax ≈27 g/L, in all cases. The more the PCC0 increased, the fewer lipids were produced. In OMW-enriched media with PCC0≈1.2 g/L, TDCW=20.9 g/L containing ≈40% w/w of lipids was recorded. In L. starkeyi cultures, when PCC0≈2.0 g/L, TDCW≈25 g/L was synthesized, whereas lipids in TDCW =24-28% w/w, similar to the experiments without OMWs, were recorded. Non-negligible dephenolization and species-dependant decolorization of the wastewater occurred. A batch-bioreactor trial by C. curvatus only with xylose (S0≈110 g/L) was performed, and TDCW=35.1 g/L (lipids in TDCW=41.3% w/w) was produced. Yeast total lipids were composed of oleic and palmitic and to lesser extent linoleic and stearic acids. C. curvatus lipids were mainly composed of non-polar fractions (i.e. triacylglycerols)

Development of a circular oriented bioprocess for microbial oil production using diversified mixed confectionery side-streams

Diversified mixed confectionery waste streams were utilized in a two-stage bioprocess to formulate a nutrient-rich fermentation media for microbial oil production. Solid-state fermentation was conducted for the production of crude enzyme consortia to be subsequently applied in hydrolytic reactions to break down starch, disaccharides, and proteins into monosaccharides, amino acids, and peptides. Crude hydrolysates were evaluated in bioconversion processes using the red yeast Rhodosporidium toruloides DSM 4444 both in batch and fed-batch mode. Under nitrogen-limiting conditions, during fed-batch cultures, the concentration of microbial lipids reached 16.6−17 g·L−1 with the intracellular content being more than 40% (w/w) in both hydrolysates applied. R. toruloides was able to metabolize mixed carbon sources without catabolite repression. The fatty acid profile of the produced lipids was altered based on the substrate employed in the bioconversion process. Microbial lipids were rich in polyunsaturated fatty acids, with oleic acid being the major fatty acid (61.7%, w/w). This study showed that mixed food side-streams could be valorized for the production of microbial oil with high unsaturation degree, pointing towards the potential to produce tailor-made lipids for specific food applications. Likewise, the proposed process conforms unequivocally to the principles of the circular economy, as the entire quantity of confectionery by-products are implemented to generate added-value compounds that will find applications in the same original industry, thus closing the loop

Attributional and consequential life cycle perspectives of second-generation polylactic acid: The benefits of integrating a recycling strategy

The climate crisis calls for a shift from petrochemicals to bio-based products to reduce environmental consequences. Polylactic acid (PLA) is one of the most widely used biopolymers, due to its mechanical properties and renewable origin, to produce bio-based compostable plastic for food packaging. The objective of this study is to determine the environmental feasibility of a second-generation PLA production based on wheat straw; and the role of a chemical recycling plant on the environmental performance of a bioproduct at an early design stage. A holistic assessment was performed through the Life Cycle Assessment (LCA) methodology considering both attributional and consequential perspectives, through a cradle-to-grave approach. The attributional LCA results show that lactic acid production was the main contributor due to the wheat straw pre-treatment and downstream separation and purification (DSP) processes. The integration of a recycling plant leads to a significant reduction of burdens, ranging from 1.38 to 0.44 kg CO2eq in the Global Warming category. Furthermore, consequential LCA results shows that the increased demand for substitute products for activities such as feeding, fertilisation and energy generation and the indirect emissions from land use change related to the conversion of land for the cultivation of raw materials are relevant factors in the environmental effects associated with the possible implementation of straw-based bioPLA production systemThis research is supported by the project Enhancing diversity in Mediterranean cereal farming systems (CerealMed), funded by PRIMA Programme and FEDER/Ministry of Science and Innovation– Spanish National Research Agency (PCI2020-111978) and the project Transition to sustainable agri-food sector bundling life cycle assessment and ecosystem services approaches (ALISE), funded by the Spanish National Research Agency (TED2021-130309B–I00). Rebolledo-Leiva R., Moreira, M.T., González-García, S. belong to the Galician Competitive Research Group (GRC ED431C-2021/37) and to the Cross-disciplinary Research in Environmental Technologies (CRETUS Research Center, ED431E 2018/01)S

Pursuing single or combined wheat straw based poly(butylene succinate) production routes: A life cycle approach of first- and second-generation feedstocks

The depletion of fossil resources and the climate change crisis call for an urgent shift to production pathways based on renewable and low-carbon sources. In addition, plastic pollution worldwide motivates the identification of new sources for their bio-based counterparts, which have an increasing demand. This research aims to evaluate the environmental feasibility of different cereal-based feedstocks for the production of poly(butylene succinate) (PBS), which is obtained from the polymerisation of succinic acid (SA) and 1,4 butanediol (BDO) monomers. The baseline scenario analysed corresponds to the use of wheat straw as a source of the fermentable sugars. Furthermore, five other cereal-based production routes combining first-generation (1G) feedstocks such as wheat and maize grain, and second-generation (2G) feedstocks, such as sorghum, barley straw, and maize stover, combined with wheat straw, were evaluated. The Life Cycle Assessment (LCA) methodology was used to identify the main hotspots of these valorisation routes at the early stage of the biorefinery design, considering all the burden categories provided by the ReCiPe impact method. The results showed that the straw-based PBS profile reached a Global Warming Potential of 3.43 kg CO2eq, whereas a range value from 2.34 to 7.27 kg CO2eq was estimated when wheat straw is combined with sorghum and barley straw, respectively. The pre-treatment stage represents a substantial impact on the strategy considered to produce fermentable sugars, particularly, for barley straw. Therefore, improvements are still required to reduce the energy demand and increase the sugar yieldThis research is supported by the project Enhancing diversity in Mediterranean cereal farming systems (CerealMed), funded by PRIMA Programme and FEDER/Ministry of Science and Innovation– Spanish National Research Agency (PCI2020-111978) and the project Transition to sustainable agri-food sector bundling life cycle assessment and ecosystem services approaches (ALISE), funded by the Spanish National Research Agency (TED2021-130309B-I00). R.R.L., M.T.M., S.G.G. belong to the Galician Competitive Research Group (GRC ED431C-2021/37) and to the Cross-disciplinary Research in Environmental Technologies (CRETUS Research Center, ED431E 2018/01)S

Valorisation of side streams from wheat milling and confectionery industries for consolidated production and extraction of microbial lipids

Crude enzymes produced via solid state fermentation (SSF) using wheat milling by-products have been employed for both fermentation media production using flour-rich waste (FRW) streams and lysis of Rhodosporidium toruloides yeast cells. Filter sterilization of crude hydrolysates was more beneficial than heat sterilization regarding yeast growth and microbial oil production. The initial carbon to free amino nitrogen ratio of crude hydrolysates was optimized (80.2 g/g) in fed-batch cultures of R. toruloides leading to a total dry weight of 61.2 g/L with microbial oil content of 61.8 % (w/w). Employing a feeding strategy where the glucose concentration was maintained in the range of 12.2 – 17.6 g/L led to the highest productivity (0.32 g/L∙h). The crude enzymes produced by SSF were utilised for yeast cell treatment leading to simultaneous release of around 80% of total lipids in the broth and production of a hydrolysate suitable as yeast extract replacement

Rhodosporidium toruloides cultivated in NaCl-enriched glucose-based media: adaptation dynamics and lipid production

In the present report and for the first time in the international literature, the impact of the addition of NaCl upon growth and lipid production on the oleaginous yeast Rhodosporidium toruloides was studied. Moreover, equally for first time, lipid production by R. toruloides was performed under non-aseptic conditions. Therefore, the potentiality of R. toruloides DSM 4444 to produce lipid in media containing several initial concentrations of NaCl with glucose employed as carbon source was studied. Preliminary batch-flask trials with increasing amounts of NaCl revealed the tolerance of the strain against NaCl content up to 6.0% (w/v). However, 4.0% (w/v) of NaCl stimulated lipid accumulation for this strain, by enhancing lipid production up to 71.3% (w/w) per dry cell weight. The same amount of NaCl was employed in pasteurized batch-flask cultures in order to investigate the role of the salt as bacterial inhibiting agent. The combination of NaCl and high glucose concentrations was found to satisfactorily suppress bacterial contamination of R. toruloides cultures under these conditions. Batch-bioreactor trials of the yeast in the same media with high glucose content (up to 150 g/L) resulted in satisfactory substrate assimilation, with almost linear kinetic profile for lipid production, regardless of the initial glucose concentration imposed. Finally, fed-batch bioreactor cultures led to the production of 37.2 g/L of biomass, accompanied by 64.5% (w/w) of lipid yield. Lipid yield per unit of glucose consumed received the very satisfactory value of 0.21 g/g, a value amongst the highest ones in the literature. The yeast lipid produced contained mainly oleic acid and to lesser extent palmitic and stearic acids, thus constituting a perfect starting material for “second generation” biodiese

Techno-economic optimization of a process superstructure for lignin valorization

Lignin, the most abundant aromatic biopolymer on Earth, is often considered a biorefinery by-product, despite its potential to be valorized into high-added-value chemicals and fuels. In this work, an integrated superstructure-based optimization model was set up and optimized using mixed-integer non-linear programming for the conversion of technical lignin to three main biobased products: aromatic monomers, phenol-formaldehyde resins, and aromatic aldehydes/acids. Several alternative conversion pathways were simultaneously compared to assess the profitability of lignins-based processes by predicting the performance of technologies with different TRL. Upon employing key technologies such as hydrothermal liquefaction, dissolution in solvent, or high-temperature electrolysis, the technical lignins could have a market value of 200 €/t when the market price for aromatic monomers, resins, and vanillin is at least 2.0, 0.8, and 15.0 €/kg, respectively. When lower product selling prices were considered, the aromatic monomers and the resins were not profitable as target products

Extraction of Phenolic Compounds from Palm Oil Processing Residues and Their Application as Antioxidants

Nusproizvodi proizvodnje palminog ulja, i to pogača od palminih sjemenki, palmina vlakna, ljuske palminih sjemenki i prazni grozdovi palminog ploda, upotrijebljeni su za ekstrakciju polifenolnih spojeva. Među tim nusproizvodima je pogača od palminih sjemenki sadržavala najviše ukupnih fenolnih spojeva, i to 5,19 mg u g suhe tvari, izraženih kao ekvivalent galne kiseline, dok je najmanje imao prazni grozd palminog ploda, i to 1,79 mg/g. Radi optimiranja ekstrakcije fenola ispitani su sljedeći parametri: vrijeme ekstrakcije i omjer tekuće i krute tvari. Najveći ukupni udjel fenola od 5,35 mg/g pri omjeru tekuće i krute tvari od 40:1 tijekom ekstrakcije od 20 min imala je pogača od palminih sjemenki. Pomoću HPLC-DAD metode određeni su glavni fenolni spojevi iz nusprodukata proizvodnje palminog ulja. Pogača od palminih sjemenki sadržavala je najviše pirogalola, te 4-hidroksibenzojeve, galne i ferulinske kiseline. Prazni grozdovi palminog ploda i palmina vlakna bili su bogati hidroksibenzojevom kiselinom, dok je pirogalol bio dominantan sastojak ekstrakta ljuski palminih sjemenki. Svi su ekstrakti imali oksidacijsku aktivnost, koja je potvrđena DPPH analizom, te ispitana dodatkom ekstrakta suncokretovom ulju radi produljenja roka trajanja. Dodatkom 0,8 % ekstrakta pogače od palminih sjemenki povećalo se indukcijsko vrijeme suncokretovog ulja za više od 50 %. Rezultati istraživanja potvrđuju da je pogača od palminih sjemenki nusproizvod s dodanom vrijednošću koji se može upotrijebiti kao antioksidans u prehrambenoj industriji.The side streams derived from the palm oil production process, namely palm kernel cake, palm pressed fibre, palm kernel shells and empty fruit bunches, were evaluated as sources of phenolic compounds. Among these streams, kernel cake had the highest total phenolic content (in mg of gallic acid equivalents (GAE) per g of dry sample) with a value of 5.19, whereas the empty fruit bunches had the lowest value (1.79). The extraction time and liquid-to-solid ratio were investigated to optimize the phenolic extraction. Kernel cake exhibited the highest total phenolic content (5.35 mg/g) with a liquid-to-solid ratio of 40:1 during 20 min of extraction. The main phenolic compounds of the extracts deriving from all byproduct streams were also identified and quantified with HPLC-DAD. Pyrogallol, 4-hydroxybenzoic acid, gallic acid and ferulic acid were the main compounds found in kernel cake extracts. Empty fruit bunch and pressed fibre extracts were also rich in 4-hydroxybenzoic acid, while pyrogallol was the predominant compound in kernel shell extracts. All extracts showed antioxidant activity as it was indicated from the results of DPPH analysis and subsequently tested in sunflower oil aiming to prolong its shelf life. The addition of 0.8 % kernel cake extract increased the induction time of sunflower oil more than 50 %. According to the results obtained in this study, kernel cake extracts could be considered as a value-added co-product with a potential application as antioxidants in the food industry