Comparison of methane yield of a novel strain of Methanothermobacter marburgensis in pure and mixed adapted culture derived from a methanation bubble column bioreactor.

The ongoing discussion regarding the use of mixed or pure cultures of hydrogenotrophic methanogenic archaea in Power-to-Methane (P2M) bioprocess applications persists, with each option presenting its own advantages and disadvantages. To address this issue, a comparison of methane (CH4) yield between a novel methanogenic archaeon belonging to the species Methanothermobacter marburgensis (strain Clermont) isolated from a biological methanation column, and the community from which it originated, was conducted. This comparison included the type strain M. marburgensis str. Marburg. The evaluation also examined how exposure to oxygen (O2) for up to 240 min impacted the CH4 yield across these cultures. While both Methanothermobacter strains exhibit comparable CH4 yield, slightly higher than that of the mixed adapted culture under non-O2-exposed conditions, strain Clermont does not display the lag time observed for strain Marburg.

Optimization of energy recovery efficiency from sweet sorghum stems by ethanol and methane fermentation processes coupling.

Taken separately, a single sweet sorghum stem bioconversion process for bioethanol and biomethane production only leads to a partial conversion of organic matter. The direct fermentation of crushed whole stem coupled with the methanization of the subsequent solid residues in a two-stage process was experimented to improve energy bioconversion yield, efficiency, and profitability. The raw stalk calorific value was 17,144.17 kJ/kg DM. Fermentation step performed using Saccharomyces cerevisiae resulted in a bioconversion yield of 261.18 g Eth/kg DM, i.e. an energy recovery efficiency of 6921.27 kJ/kg DM. The methanogenic potentials were 279 and 256 LCH4/kg DM, respectively, for raw stem and fermentation residues, i.e. energy yields of 10,013.31 and 9187.84 kJ/kg DM, respectively. Coupling processes have significantly increased yield and made it possible to reach 13,309.57 kJ/kg DM, i.e. 77.63% of raw stem energy recovery yield, compared to 40.37% and 58.40%, respectively, for single fermentation and methanization processes.

Sweet sorghum stem is a viable feedstock source for efficient coproduction of ethanol and methaneSorghum stems calorific value determination revealed an energy potential of 17.15 MJ/kg DMEnergy recovery by single methanization yielded 18.03% more than ethanol fermentationCoupling processes has significantly increased energy recovery yield and profitability.

Bioinformatics Modelling and Metabolic Engineering of the Branched Chain Amino Acid Pathway for Specific Production of Mycosubtilin Isoforms in Bacillus subtilis.

Mycosubtilin belongs to the family of lipopeptides. Different isoforms with various antifungal activities can be obtained according to the length and the isomery of the fatty acid. In this work, the activities of the mycosubtilin isoforms were first studied against the pathogen Aspergillus niger, revealing the high activity of the anteiso-C17 isoform. Modification of the mycosubtilin isoform patterns during cultures of the natural strain Bacillus subtilis ATCC 6633 was then investigated through amino acid feeding experiments. In parallel, single-gene knockouts and single-gene overexpression, leading to the overproduction of the anteiso-C15 fatty acid chains, were predicted using informatics tools which provide logical reasoning with formal models of reaction networks. In this way, it was in silico predicted that the single overexpression of the ilvA gene as well as the single knockout of the codY gene may lead to the overproduction of anteiso-C15 fatty acid chains. For the first time, it has been demonstrated that overexpression of ilvA helps to enhance the furniture of odd anteiso fatty acids leading to a favored mycosubtilin anteiso-C17 production pattern (+41%). Alternatively, a knock-out codY mutant led to a higher furniture of even iso fatty acids, leading to a favored mycosubtilin iso-C16 production pattern (+180%). These results showed that increased selective synthesis of particular isoforms of mycosubtilin through metabolic engineering is feasible, disclosing the interest of these approaches for future development of lipopeptide-producing strains.

Bioproducts generation from carboxylate platforms by the non-conventional yeast Yarrowia lipolytica.

In recent years, there has been a growing interest in the use of renewable sources for bio-based production aiming at developing sustainable and feasible approaches towards a circular economy. Among these renewable sources, organic wastes (OWs) can be anaerobically digested to generate carboxylates like volatile fatty acids (VFAs), lactic acid, and longer-chain fatty acids that are regarded as novel building blocks for the synthesis of value-added compounds by yeasts. This review discusses on the processes that can be used to create valuable molecules from OW-derived VFAs; the pathways employed by the oleaginous yeast Yarrowia lipolytica to directly metabolize such molecules; and the relationship between OW composition, anaerobic digestion, and VFA profiles. The review also summarizes the current knowledge about VFA toxicity, the pathways by which VFAs are metabolized and the metabolic engineering strategies that can be employed in Y. lipolytica to produce value-added biobased compounds from VFAs.

Optimization of limonene biotransformation for the production of bulk amounts of α-terpineol.

The biotransformation of R-(+)-limonene into high concentrations of R-(+)-α-terpineol by Sphingobium sp. was investigated in order to optimize the main process variables (pH, biocatalyst concentration, substrate concentration, temperature and agitation). This strategy comprised the screening of variables by a Plackett-Burman design followed by a Central Composite Design. The statistical analysis showed that the optimal α-terpineol production were at 28 °C and pH 7.0, with a limonene concentration of 350 g/L of organic phase agitation of 200 rpm and a biocatalyst concentration of 2.8 g/L of aqueous phase (OD600 = 8). Further trials showed that the R-(+)-α-terpineol concentration was higher (240 g/L after 96 h) when using a ratio of 1:3 (v.v-1) of organic:aqueous phases. However, the total production and yield (in terms of biomass) of α-terpineol would be maximized for an aqueous:organic ratio of 1:1. The experimental design optimization adopted herein was an effective tool for this type of study.

Hydrogen production by dark fermentation from pre-fermented depackaging food wastes.

In this study, a specific fraction of food waste, i.e. depackaging waste, was studied as substrate for hydrogen production by dark fermentation. During storage and transport of this liquid mixture, inhibitory compounds like acids or alcohol might be produced by endogenous flora. A factorial fractional design based on the composition of the substrate was used to determine the best condition to convert this substrate into hydrogen. First results indicated that the consortium used might convert high quantity of lactate into hydrogen. A batch culture confirmed that lactate was used as the main carbon source and a global yield of 0.4molH2·mollactate-1 was obtained. This study demonstrated the ability of the consortium tested to convert different carbon sources (carbohydrates or lactate) with good efficiency. These data represented an important parameter in the prospect of using an industrial substrate whose composition is liable to vary according to the conditions of storage and transport.

Cloning and Characterization of the Gene Encoding Alpha-Pinene Oxide Lyase Enzyme (Prα-POL) from Pseudomonas rhodesiae CIP 107491 and Production of the Recombinant Protein in Escherichia coli.

The alpha-pinene oxide lyase (Prα-POL) from Pseudomonas rhodesiae CIP107491 belongs to catabolic alpha-pinene degradation pathway. In this study, the gene encoding Prα-POL has been identified using mapping approach combined to inverse PCR (iPCR) strategy. The Prα-POL gene included a 609-bp open reading frame encoding 202 amino acids and giving rise to a 23.7 kDa protein, with a theoretical isoelectric point (pI) of 5.23. The amino acids sequence analysis showed homologies with those of proteins with unknown function from GammaProteobacteria group. Identification of a conserved domain in amino acid in positions 18 to 190 permitted to classify Prα-POL among the nuclear transport factor 2 (NTF2) protein superfamily. Heterologous expression of Prα-POL, both under its native form and with a histidin tag, was successfully performed in Escherichia coli, and enzymatic kinetics were analyzed. Bioconversion assay using recombinant E. coli strain allowed to reach a rate of isonovalal production per gramme of biomass about 40-fold higher than the rate obtained with P. rhodesiae.

Improvement and modeling of culture parameters to enhance biomass and lipid production by the oleaginous yeast Cryptococcus curvatus grown on acetate.

The improvement of culture parameters for lipid production from acetate as carbon source was investigated using the oleaginous yeast Cryptococcus curvatus. A new pH regulation system dispensing acetate was developed for fed-batch culture and allowed obtaining nearly 80 g/L biomass within 60 h with a maximal growth rate of 0.28 h(-1). A biological model was developed from experimental data. The influence of three C/N ratios of 300, 500 and 900 were tested during a multi-phases process on lipid accumulation. The C/N ratio of 300 was reported to be the most suitable for lipid storage. No significant increase of lipids content was obtained with higher value. A maximal content of 60% DCW of lipid was obtained. The determination of fatty acids profiles of the microbial oils has confirmed that the valorization of acetate by microbial oils production was a promising perspective.

Biological upgrading of volatile fatty acids, key intermediates for the valorization of biowaste through dark anaerobic fermentation.

VFAs can be obtained from lignocellulosic agro-industrial wastes, sludge, and various biodegradable organic wastes as key intermediates through dark fermentation processes and synthesized through chemical route also. They are building blocks of several organic compounds viz. alcohol, aldehyde, ketones, esters and olefins. These can serve as alternate carbon source for microbial biolipid, biohydrogen, microbial fuel cells productions, methanisation, and for denitrification. Organic wastes are the substrate for VFA platform that is of zero or even negative cost, giving VFA as intermediate product but their separation from the fermentation broth is still a challenge; however, several separation technologies have been developed, membrane separation being the most suitable one. These aspects will be reviewed and results obtained during anaerobic treatment of slaughterhouse wastes with further utilisation of volatile fatty acids for yeast cultivation have been discussed.

Bioconversion of volatile fatty acids into lipids by the oleaginous yeast Yarrowia lipolytica.

The valorization of volatile fatty acids into microbial lipids by the oleaginous yeast Yarrowia lipolytica was investigated. Therefore, a two-stage fed-batch strategy was designed: the yeast was initially grown on glucose or glycerol as carbon source, then sequential additions of acetic acid under nitrogen limiting conditions were performed after glucose or glycerol exhaustion. The typical values obtained with an initial 40 g/L concentration of glucose were close to 31 g/L biomass, a lipid concentration of 12.4 g/L, which correspond to a lipid content of the biomass close to 40%. This cultivation strategy was also efficient with other volatile fatty acids (butyric and propionic acids) or with a mixture of these three VFAs. The lipids composition was found quite similar to that of vegetable oils. The study demonstrated the feasibility of simultaneous biovalorization of volatile fatty acids and glycerol, two cheap industrial by-products.

Recent developments in microbial oils production: a possible alternative to vegetable oils for biodiesel without competition with human food?

Since centuries vegetable oils are consumed as human food but it also finds applications in biodiesel production which is attracting more attention. But due to being in competition with food it could not be sustainable and leads the need to search for alternative. Nowdays microbes-derived oils (single cell oils) seem to be alternatives for biodiesel production due to their similar composition to that of vegetable oils. However, the cold flow properties of the biodiesel produced from microbial oils are unacceptable and have to be modified by an efficient transesterification. Glycerol which is by product of transesterification can be valorised into some more useful products so that it can also be utilised along with biodiesel to simplify the downstream processing. The review paper discusses about various potent microorganisms for biodiesel production, enzymes involved in the lipid accumulation, lipid quantification methods, catalysts used in transesterification (including enzymatic catalyst) and valorisation of glycerol.

Fed-batch production of gluconic acid by terpene-treated Aspergillus niger spores.

Aspergillus niger spores were used as catalyst in the bioconversion of glucose to gluconic acid. Spores produced by solid-state fermentation were treated with 15 different terpenes including monoterpenes and monoterpenoids to permeabilize and inhibit spore germination. It was found that spore membrane permeability is significantly increased by treatment with terpenoids when compared to monoterpenes. Best results were obtained with citral and isonovalal. Studies were carried out to optimize spores concentration (10(7)-10(10) spores/mL), terpene concentrations in the bioconversion medium and time of exposure (1-18 h) needed for permeabilization of spores. Fed-batch production of gluconate was done in a bioreactor with the best conditions [10(9) spores/mL of freeze-thawed spores treated with citral (3% v/v) for 5 h] followed by sequential additions of glucose powder and pH-regulated with a solution containing 2 mol/L of either NaOH or KOH. Bioconversion performance of the spore enzyme was compared with the commercial glucose oxidase at 50, 60, and 70 degrees C. Results showed that the spore enzyme was comparatively stable at 60 degrees C. It was also found that the spores could be reutilized for more than 14 cycles with almost similar reaction rate. Similar biocatalytic activity was rendered by spores even after its storage of 1 year at -20 degrees C. This study provided an experimental evidence of the significant catalytic role played by A. niger spore in bioconversion of glucose to gluconic acid with high yield and stability, giving protection to glucose oxidase.

Permeabilization and inhibition of the germination of spores of Aspergillus niger for gluconic acid production from glucose.

In this study, the role of citral to permeabilize the spores of Aspergillus niger and replace sodium azide in the bioconversion medium was studied. Further, characterization of glucose oxidase of spores was carried out by exposing both permeabilized and unpermeabilized spores to different pressures (1, 2, 2.7 kb) and temperatures (60, 70, 80, 90 degrees C). Unpermeabilized spores after exposure to high temperatures were permeabilized by freezing before using as catalyst in the bioconversion reaction. Results showed that citral permeabilized the spores and could inhibit spore germination in the bioconversion medium. Rate of reaction was significantly increased from 1.5 to 4.35 g/Lh which was higher than the commercial glucose oxidase 2g/Lh). Glucose oxidase activity of A. niger was resistant to pressure. However, pressure treatment could not permeabilize them. Behaviour of fresh and permeabilized spores to temperature varied significantly. Glucose oxidase activity of fresh spores exposed to high temperature was unaffected at 70 degrees C till 15 min and 84% of relative activity was retained even after 1h at 70 degrees C while permeabilized spore got inactivated at 70 degrees C for 15 min, which followed the same pattern as commercial glucose oxidase. Cellular membrane integrity was lost due to permeabilization by freezing which resulted in heat-inactivation of glucose oxidase when spores were permeabilized before heat treatment. Thus, glucose oxidase of spore remains heat stable when unpermeabilized and active while permeabilized and its reaction rate is higher than the commercial glucose oxidase.

Production of trans-2-methyl-5-isopropylhexa-2,5-dienoic acid by Pseudomonas rhodesiae CIP 107491.

The feasibility of trans-2-methyl-5-isopropylhexa-2,5-dienoic acid (novalic acid) accumulation using the alpha-pinene degradation pathway of Pseudomonas rhodesiae CIP 107491 was studied. This appeared possible by using concentrated living bacterial cells produced under oxygen limitation with alpha-pinene as sole carbon source. The second step of the process, the bioconversion itself, had to be performed without oxygen limitation due to the need for cofactor regeneration. Results showed that a not yet reported cofactor-dependent enzymatic isomerization of isonovalal into novalal was likely to occur and that both aldehyde isomers could be oxidized to the corresponding acid. Precursors tested, alpha-pinene oxide and isonovalal had a strong permeabilization effect on bacterial cells. This effect, which increased from the oxide to the aldehyde, led to an inactivation of the respiratory chain and to acids synthesis stop. Present results allowed to obtain about 12 g/L acids (80% novalic acid) with an average yield close to 50% after 12h reaction in a biphasic system using alpha-pinene oxide as precursor .