Microbial conversion of lignin rich biomass hydrolysates to medium chain length polyhydroxyalkanoates (mcl-PHA) using Pseudomonas putida KT2440.

As world moves toward increasing number of products being produced from renewable lignocellulosic agricultural and forest residues, the major classes of products that will shift to greener routes on priority are energy, fuels, and materials in that order. In materials segment, polyhydroxyalkanoates are an emerging class of biopolyesters with several potential industrial uses. The present work investigates medium chain length polyhydroxyalkanoates (mcl-PHA) producing capabilities of Pseudomonas putida KT2440 from a mixture of compounds produced from lignocellulosic biomass deconstruction. The hydrolysates obtained from nitric acid pretreatment of lignin rich cotton stalk (CS) and palm empty fruit bunch (EFB) were used as substrates for production of mcl-PHA. Presence of 3-hydroxydecanoate and 3-hydroxyocytanoate observed on GC-MS confirmed PHA accumulation in the cells. PHA accumulation was estimated between 20% and 35% of cell dry weight when grown on both model substrates as well as biomass hydrolysates. PHA titers obtained on hydrolysates of CS and EFB were 0.24 g/L and 0.21 g/L, respectively.

Recent progress in the microbial production of xylonic acid.

Interest in the production of renewable chemicals from biomass has increased in the past years. Among these chemicals, carboxylic acids represent a significant part of the most desirable bio-based products. Xylonic acid is a five-carbon sugar-acid obtained from xylose oxidation that can be used in several industrial applications, including food, pharmaceutical, and construction industries. So far, the production of xylonic acid has not yet been available at an industrial scale; however, several microbial bio-based production processes are under development. This review summarizes the recent advances in pathway characterization, genetic engineering, and fermentative strategies to improve xylonic acid production by microorganisms from xylose or lignocellulosic hydrolysates. In addition, the strengths of the available microbial strains and processes and the major requirements for achieving biotechnological production of xylonic acid at a commercial scale are discussed. Efficient native and engineered microbial strains have been reported. Xylonic acid titers as high as 586 and 171 g L-1 were obtained from bacterial and yeast strains, respectively, in a laboratory medium. Furthermore, relevant academic and industrial players associated with xylonic acid production will be presented.

Oleaginous yeasts- substrate preference and lipid productivity: a view on the performance of microbial lipid producers.

BACKGROUND: Oleaginous yeasts are promising microbial platforms for sustainable, bio-based production of biofuels and oleochemical building blocks. Bio-based residues provide sustainable and cost-effective carbon sources for fermentative yeast oil production without land-use change. Considering the regional abundancy of different waste streams, we chose complex biomass residue streams of marine origin; macroalgae hydrolysate, and terrestrial origin; wheat straw hydrolysate in the presence, and absence of corn steep liquor as a complex nitrogen source. We investigated the biomass and lipid yields of an array of well-described oleaginous yeasts; R. glutinis, T. asahii, R. mucilaginosa, R. toruloides, C. oleaginosus growing on these hydrolysates. Furthermore, their sugar utilization, fatty acid profile, and inhibitory effect of the hydrolysates on yeast growth were compared. For correlative reference, we initially performed comparative growth experiments for the strains on individual monomeric sugars separately. Each of these monomeric sugars was a dominant carbon source in the complex biomass hydrolysates evaluated in this study. In addition, we evaluated N-acetylglucosamine, the monomeric building block of chitin, as a low-cost nitrogen and carbon source in yeast fermentation. RESULTS: C. oleaginosus provided the highest biomass and lipid yields. In the wheat straw and brown algae hydrolysates, this yeast strain gained 7.5 g/L and 3.8 g/L lipids, respectively. Cultivation in algae hydrolysate resulted in a higher level of unsaturated fatty acids in the lipids accumulated by all yeast strains. R. toruloides and C. oleaginosus were able to effectively co-utilize mannitol, glucose, and xylose. Growth rates on wheat straw hydrolysate were enhanced in presence of corn steep liquor. CONCLUSIONS: Among the yeast strains investigated in this study, C. oleaginosus proved to be the most versatile strain in terms of substrate utilization, productivity, and tolerance in the complex media. Various fatty acid profiles obtained on each substrate encourage the manipulation of culture conditions to achieve the desired fatty acid composition for each application. This could be accomplished by combining the element of carbon source with other formerly studied factors such as temperature and oxygen. Moreover, corn steep liquor showed promise for enhancement of growth in the oleaginous strains provided that carbon substrate is available.

Chromosomal mutations in Escherichia coli that improve tolerance to nonvolatile side-products from dilute acid treatment of sugarcane bagasse.

Lignocellulosic biomass provides attractive nonfood carbohydrates for the production of ethanol, and dilute acid pretreatment is a biomass-independent process for access to these carbohydrates. However, this pretreatment also releases volatile and nonvolatile inhibitors of fermenting microorganisms. To identify unique gene products contributing to sensitivity/tolerance to nonvolatile inhibitors, ethanologenic Escherichia coli strain LY180 was adapted for growth in vacuum-treated sugarcane bagasse acid hydrolysate (VBHz) lacking furfural and other volatile inhibitors. A mutant, strain AQ15, obtained after approximately 500 generations of growth in VBHz, grew and fermented the sugars in a medium with 50% VBHz. Comparative genome sequence analysis of strains AQ15 and LY180 revealed 95 mutations in strain AQ15. Six of these mutations were also found in strain SL112, an independent inhibitor-tolerant derivative of strain LY180. Among these six mutations, null mutations in mdh and bacA were identified as contributing factors to VBHz tolerance in strain AQ15, based on the genetic and physiological analysis. The deletion of either gene in strain LY180 increased tolerance to VBHz from approximately 30-50% (vol/vol). Considering the location and physiological role of the two enzymes in the cell, it is likely that the two enzymes contribute to the VBHz sensitivity of ethanologenic E. coli by different mechanisms.

Xylitol production on sugarcane biomass hydrolysate by newly identified Candida tropicalis JA2 strain.

Xylitol is a building block for a variety of chemical commodities, besides being widely used as a sugar substitute in the food and pharmaceutical industries. The aim of this work was to develop a microbial process for xylitol production using sugarcane bagasse hydrolysate as substrate. In this context, 218 non-Saccharomyces yeast strains were screened by growth on steam-exploded sugarcane bagasse hydrolysate containing a high concentration of acetic acid (8.0 g/L). Seven new Candida tropicalis strains were selected and identified, and their ability to produce xylitol on hydrolysate at low pH (4.6) under aerobic conditions was evaluated. The most efficient strain, designated C. tropicalis JA2, was capable of producing xylitol with a yield of 0.47 g/g of consumed xylose. To improve xylitol production by C. tropicalis JA2, a series of experimental procedures were employed to optimize pH and temperature conditions, as well as nutrient source, and initial xylose and inoculum concentrations. C. tropicalis JA2 was able to produce 109.5 g/L of xylitol with a yield of 0.86 g/g of consumed xylose, and with a productivity of 2.81 g·L·h, on sugarcane bagasse hydrolysate containing 8.0 g/L acetic acid and177 g/L xylose, supplemented with 2.0 g/L yeast nitrogen base and 4.0 g/L urea. Thus, it was possible to identify a new C. tropicalis strain and to optimize the xylitol production process using sugarcane bagasse hydrolysate as a substrate. The xylitol yield on biomass hydrolysate containing a high concentration of acetic acidobtained in here is among the best reported in the literature.

Influence of genetic background of engineered xylose-fermenting industrial Saccharomyces cerevisiae strains for ethanol production from lignocellulosic hydrolysates.

An industrial ethanol-producing Saccharomyces cerevisiae strain with genes of fungal oxido-reductive pathway needed for xylose fermentation integrated into its genome (YRH1415) was used to obtain haploids and diploid isogenic strains. The isogenic strains were more effective in metabolizing xylose than YRH1415 strain and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting from the hydrolysis of lignocellulosic biomass (switchgrass). The rate of xylose consumption did not appear to be affected by the ploidy of strains or the presence of two copies of the xylose fermentation genes but by heterozygosity of alleles for xylose metabolism in YRH1415. Furthermore, inhibitor tolerance was influenced by the heterozygous genome of the industrial strain, which also showed a marked influenced on tolerance to increasing concentrations of toxic compounds, such as furfural. In this work, selection of haploid derivatives was found to be a useful strategy to develop efficient xylose-fermenting industrial yeast strains.

Pichia anomala 29X: a resistant strain for lignocellulosic biomass hydrolysate fermentation.

To efficiently use lignocellulosic biomass hydrolysates as fermentation media for bioethanol production, besides being capable of producing significant amount of ethanol, the fermenting host should also meet the following two requirements: (1) resistant to the inhibitory compounds formed during biomass pretreatment process, (2) capable of utilizing C5 sugars, such as xylose, as carbon source. In our laboratory, a screening was conducted on microorganisms collected from environmental sources for their tolerance to hydrolysate inhibitors. A unique resistant strain was selected and identified as Pichia anomala (Wickerhamomyces anomalus), deposited as CBS 132101. The strain is able to produce ethanol in various biomass hydrolysates, both with and without oxygen. Besides, the strain could assimilate xylose and use nitrate as N source. These physiological characteristics make P. anomala an interesting strain for bioethanol production from lignocellulosic biomass hydrolysates.

Physicochemical Pretreatment of Vietnamosasa pusilla for Bioethanol and Xylitol Production.

The consumption of fossil fuels has resulted in severe environmental consequences, including greenhouse gas emissions and climate change. Therefore, transitioning to alternative energy sources, such as cellulosic ethanol, is a promising strategy for reducing environmental impacts and promoting sustainable low-carbon energy. Vietnamosasa pusilla, an invasive weed, has been recognized as a high potential feedstock for sugar-based biorefineries due to its high total carbohydrate content, including glucan (48.1 ± 0.3%) and xylan (19.2 ± 0.4%). This study aimed to examine the impact of NaOH pretreatment-assisted autoclaving on V. pusilla feedstock. The V. pusilla enzymatic hydrolysate was used as a substrate for bioethanol and xylitol synthesis. After treating the feedstock with varying concentrations of NaOH at different temperatures, the glucose and xylose recovery yields were substantially higher than those of the untreated material. The hydrolysate generated by enzymatic hydrolysis was fermented into bioethanol using Saccharomyces cerevisiae TISTR 5339. The liquid byproduct of ethanol production was utilized by Candida tropicalis TISTR 5171 to generate xylitol. The results of this study indicate that the six- and five-carbon sugars of V. pusilla biomass have great potential for the production of two value-added products (bioethanol and xylitol).

Improved biohydrogen production via graphene oxide supported granular system based on algal hydrolyzate, secondary sewage sludge and bacterial consortia.

Biohydrogen production using renewable sources has been regarded as one of the most sustainable ways to develop low-cost and green production technology. In order to achieve this objective, herein biohydrogen production has been conducted using the combination of untreated secondary sewage sludge (Sss), algal biomass hydrolyzate (Abh), graphene oxide (GO) and bacterial consortia that forms a granular system. Thus, naturally formed granular system produced cumulative H2 of 1520 mL/L in 168 h with the maximum production rate of 13.4 mL/L/h in 96 h at initial pH 7.0, and optimum temperature of 37 °C. It is noticed that the combination of Abh, Sss and GO governed medium showed 42.05 % higher cumulative H2 production along with 22.71 % higher production rate as compared to Abh and Sss based H2 production medium. The strategy presented herein may find potential applications for the low-cost biohydrogen production using waste biomasses including Sss and Abh.

Corrigendum: Corynebacterium glutamicum as an efficient omnivorous microbial host for the bioconversion of lignocellulosic biomass.

[This corrects the article DOI: 10.3389/fbioe.2022.827386.].

Corynebacterium glutamicum as an Efficient Omnivorous Microbial Host for the Bioconversion of Lignocellulosic Biomass.

Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate lignin-derived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, 13C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of ß-ketoadipate pathway in C. glutamicum. 13C-fingerprinting in the presence of both glucose and aromatics also revealed coumarate to be the most preferred aromatic by C. glutamicum contributing 74 and 59% of its carbon for the synthesis of glutamate and aspartate respectively. 13C-fingerprinting also confirmed the activity of ortho-cleavage pathway, anaplerotic pathway, and cataplerotic pathways. Finally, the engineered C. glutamicum strain grew well in biomass hydrolysate containing pentose and hexose sugars and produced L-lactate at a concentration of 47.9 g/L and a yield of 0.639 g/g from sugars with simultaneous utilization of aromatics. Succinate and acetate co-products were produced at concentrations of 8.9 g/L and 3.2 g/L, respectively. Our findings open the door to valorize all the major carbon components of biomass hydrolysate by using C. glutamicum as a microbial host for biomanufacturing.

Synthesis of polyacrylamide/polystyrene interpenetrating polymer networks and the effect of textural properties on adsorption performance of fermentation inhibitors from sugarcane bagasse hydrolysate.

Economical removal of fermentation inhibitors from lignocellulosic hydrolysate plays a considerable role in bioconversion of lignocellulose biomass. In this work, the textural properties of polyacrylamide/polystyrene interpenetrating polymer networks (PAM/PS IPNs) on adsorption of fermentation inhibitors from sugarcane bagasse hydrolysate (SCBH) were investigated for the first time. The results showed that, the specific surface area, pore diameter and surface polarity had important influence on its adsorption performance towards sugars, organic acids, furans and acid-soluble lignin. The PAM/PS IPNs under the optimal copolymerization situation achieved the high selectivity coefficients of 4.07, 14.9, 21.2 and 25.8 with respective to levulinic acid, furfural, hydroxymethylfurfural (HMF) and acid-soluble lignin, and had a low total sugar loss of 2.09%. Overall, this research puts forward a design and synthetic strategy for adsorbent to remove fermentation inhibitors from lignocellulosic hydrolysate.

Tolerance and metabolic response of Pseudomonas taiwanensis VLB120 towards biomass hydrolysate-derived inhibitors.

BACKGROUND: Bio-conversion of lignocellulosic biomass to high-value products offers numerous benefits; however, its development is hampered by chemical inhibitors generated during the pretreatment process. A better understanding of how microbes naturally respond to those inhibitors is valuable in the process of designing microorganisms with improved tolerance. Pseudomonas taiwanensis VLB120 is a natively tolerant strain that utilizes a wide range of carbon sources including pentose and hexose sugars. To this end, we investigated the tolerance and metabolic response of P. taiwanensis VLB120 towards biomass hydrolysate-derived inhibitors including organic acids (acetic acid, formic acid, and levulinic acid), furans (furfural, 5-hydroxymethylfurfural), and phenols (vanillin). RESULTS: The inhibitory effect of the tested compounds varied with respect to lag phase, specific growth rate, and biomass yield compared to the control cultures grown under the same conditions without addition of inhibitors. However, P. taiwanensis was able to oxidize vanillin and furfural to vanillic acid and 2-furoic acid, respectively. Vanillic acid was further metabolized, whereas 2-furoic acid was secreted outside the cells and remained in the fermentation broth without further conversion. Acetic acid and formic acid were completely consumed from the fermentation broth, while concentration of levulinic acid remained constant throughout the fermentation process. Analysis of free intracellular metabolites revealed varying levels when P. taiwanensis VLB120 was exposed to inhibitory compounds. This resulted in increased levels of ATP to export inhibitors from the cell and NADPH/NADP ratio that provides reducing power to deal with the oxidative stress caused by the inhibitors. Thus, adequate supply of these metabolites is essential for the survival and reproduction of P. taiwanensis in the presence of biomass-derived inhibitors. CONCLUSIONS: In this study, the tolerance and metabolic response of P. taiwanensis VLB120 to biomass hydrolysate-derived inhibitors was investigated. P. taiwanensis VLB120 showed high tolerance towards biomass hydrolysate-derived inhibitors compared to most wild-type microbes reported in the literature. It adopts different resistance mechanisms, including detoxification, efflux, and repair, which require additional energy and resources. Thus, targeting redox and energy metabolism in strain engineering may be a successful strategy to overcome inhibition during biomass hydrolysate conversion and lead to development of more robust strains.

Rediscovering Acetate Metabolism: Its Potential Sources and Utilization for Biobased Transformation into Value-Added Chemicals.

One of the great advantages of microbial fermentation is the capacity to convert various carbon compounds into value-added chemicals. In this regard, there have been many efforts to engineer microorganisms to facilitate utilization of abundant carbon sources. Recently, the potential of acetate as a feedstock has been discovered; efforts have been made to produce various biochemicals from acetate based on understanding of its metabolism. In this review, we discuss the potential sources of acetate and summarized the recent progress to improve acetate utilization with microorganisms. Furthermore, we also describe representative studies that engineered microorganisms for the production of biochemicals from acetate.

Furfural production from biomass pretreatment hydrolysate using vapor-releasing reactor system.

Biomass hydrolysate from autohydrolysis pretreatment was used for furfural production considering it is in rich of xylose, xylo-oligomers, and other decomposition products from hemicellulose structure. By using the vapor-releasing reactor system, furfural was protected from degradation by separating it from the reaction media. The maximum furfural yield of 73% was achieved at 200 °C for biomass hydrolysate without the use of the catalyst. This is because the presence of organic acids such as acetic acid in hydrolysate functioned as a catalyst. According to the results in this study, biomass hydrolysate with a vapor-releasing system proves to be efficient for furfural production. The biorefinery process which allows the separation of xylose-rich autohydrolysate from other parts from biomass feedstock also improves the overall application of the biomass.

Valorisation of tuna processing waste biomass: isolation, purification and characterisation of four novel antioxidant peptides from tuna by-product hydrolysate.

Tuna protein hydrolysate (TPH) was prepared by hydrolysis with Prolyve BS and fractionated by membranes process. The antioxidant activities of recovered peptide fractions were evaluated. Four novel antioxidant peptides that were isolated from nanofiltration retentate exhibited the highest antioxidant activity, using gel chromatography and reversed phase high-performance liquid chromatography. The amino acid sequences of isolated peptides were identified as Tyr-Glu-Asn-Gly-Gly (P2), Glu-Gly-Tyr-Pro-Trp-Asn (P4), Tyr-Ile-Val-Tyr-Pro-Gly (P7) and Trp-Gly-Asp-Ala-Gly-Gly-Tyr-Tyr (P8) with molecular weights of 538.46, 764.75, 710.78 and 887.85 Da, respectively. P2, P4, P7 and P8 exhibited good scavenging activities on hydroxyl radical (IC50 0.41, 0.327, 0.17 and 0.042 mg/ml), DPPH radical (IC50 0.666, 0.326, 0.451 and 0.377 mg/ml) and superoxide radical (IC50 0.536, 0.307, 0.357 and 0.115 mg/ml). P7 was effective against lipid peroxidation in the model system. The isolated peptides might be useful used as natural food additive in food industry and formulation of nutritional products.

Improvement of butanol production from a hardwood hemicelluloses hydrolysate by combined sugar concentration and phenols removal.

The feasibility of using hardwood hemicellulosic pre-hydrolysate recovered from a dissolving pulping process for Acetone-Butanol-Ethanol (ABE) fermentation has been investigated. Dilutions and detoxification methods based on flocculation and nanofiltration were tested to determine the inhibitory concentration of phenolic compounds and to evaluate the efficiency of inhibitors removal on fermentation. Flocculation carried out with ferric sulfate was the most effective method for removal of phenolics (56%) and acetic acid (80%). The impact on fermentation was significant, with an ABE production of 6.40 g/L and 4.25 g/L when using flocculation or combined nanofiltration/flocculation respectively, as compared to a non-significant production for the untreated hydrolysate. By decreasing the toxicity effect of inhibitors, this study reports for the first time that the use of these techniques is efficient to increase the inhibitory concentration threshold of phenols, from 0.3g/L in untreated hydrolysate, to 1.1g/L in flocculated and in nanofiltrated and flocculated hydrolysates.

Enhanced succinic acid production and magnesium utilization by overexpression of magnesium transporter mgtA in Escherichia coli mutant.

In this study, a novel engineered Escherichia coli strain KMG111 was constructed by overexpression of mgtA in E. coli mutant DC1515. By adopting KMG111, nearly a concentration of succinic acid (32.41gL(-1)) with a yield of 0.81gg(-1) glucose, could be obtained in a batch fermentation by using the low-cost mixture of Mg(OH)2 and NH3·H2O to replace MgCO3 as the alkaline neutralizer. Moreover, the effect of the inhibitory compounds in lignocellulosic hydrolyzates on cell growth and succinic acid production could be relieved. In a 3-L bioreactor, the overall productivity and yield of succinic acid in the whole anaerobic stage were 2.15gL(-1)h(-1) and 0.86gg(-1) total sugar, respectively. This study was the first to report decreased alkaline neutralizer cost via genetic manipulation for succinic acid production, which contributed to the industrialization of this microbial synthesis process.