Hydrolysis Fermentation Research Articles

Engineering a xylose fermenting yeast for lignocellulosic ethanol production.

Lignocellulosic ethanol is produced by yeast fermentation of lignocellulosic hydrolysates generated by chemical pretreatment and enzymatic hydrolysis of plant cell walls. The conversion of xylose into ethanol in hydrolysates containing microbial inhibitors is a major bottleneck in biofuel production. We identified sodium salts as the primary yeast inhibitors, and evolved a Saccharomyces cerevisiae strain overexpressing xylose catabolism genes in xylose or glucose-mixed medium containing sodium salts. The fully evolved yeast strain can efficiently convert xylose in the hydrolysates to ethanol on an industrial scale. We elucidated that the amplification of xylA, XKS1 and pentose phosphate pathway-related genes TAL1, RPE1, TKL1, RKI1, along with mutations in NFS1, TRK1, SSK1, PUF2 and IRA1, are responsible and sufficient for the effective xylose utilization in corn stover hydrolysates containing high sodium salts. Our evolved or reverse-engineered yeast strains enable industrial-scale production of lignocellulosic ethanol and the genetic foundation we uncovered can also facilitate transfer of the phenotype to yeast cell factories producing chemicals beyond ethanol.

Exploring the Potential of Nyonya Kuih Residues as the Substrate for Yeast Cell Protein Production

Nyonya kuih, a popular Malaysian traditional cuisine that have short shelf life due to their high moisture and fat contents. The unsold kuihs usually ended up as food residue. Thus, kuih talam(KT) and kuih lapis (KL), two varieties of Nyonya kuih were chosen as the investigative substrates to explore the recycling potential of this food residue. Initially, the effect of fat content on the enzymatic hydrolysability of the residues to release fermentable sugar was investigated. The efficiency of lipase-pretreatment (LP), polyvinylpyrrolidone-post-treatment (PVP) and their combination in lipid removal was determined. Then, the potential of the resulting glucose-rich hydrolysates for Saccharomyces cerevisiae yeast cell biomass production was assessed, with molasses as the positive control. Results indicated that combined LP and PVP (LP-PVP) significantly (p<0.05) reduced crude fat content, while increasing the total sugar content of KT and KL hydrolysates by approximately 3.4 and 26.3 times, respectively. Yeast cell biomass produced from KT and KL hydrolysates were increased by 23% and 39%, respectively after LP-PVP treatment. Despite the yeast cells’ yield (by dry cell weight) from molasses fermentation being 19% and 11% higher than those from KT and KL hydrolysates, respectively, KT and KL hydrolysates were proven superior due to the significantly higher (p<0.05) crude protein content in yeast cell produced from both hydrolysates. Further study is needed to enhance yeast cell yield from the fermentation of KT and KL hydrolysates, thereby improving the overall protein yield from the fermentation process. This study provides new insight into a novel food residue recycle strategy, promoting sustainable food production aligned with SDG 12.

Enhancement of hydrogen production in dark fermentation of corn stover hydrolysates through composite electric field pretreatment: Improving enzymatic efficiency and regulating microbial metabolic balance

This study investigates the effects of composite electric field pretreatment (CEP) on hydrogen production from corn stover enzymatic hydrolysates in dark fermentation. The findings reveal that under optimal conditions, the CEP group achieved a cumulative hydrogen yield of 77.3 ± 2.6 mL/g TS, marking a 55.3 % increase compared to the control group without the electric field. CEP significantly enhances the fermentation capacity of enzymatic hydrolysates during the acid-stage of dark fermentation by disrupting the lignin structure and optimizing cellulase hydrolysis efficiency. Additionally, pH self-regulation is facilitated through the interaction between volatile fatty acids (VFAs) and ammonia nitrogen. Microbial community analysis revealed that CEP shifts the metabolic balance between Clostridium_sensu_stricto_1 and Lactococcus, leading to increased hydrogen yield and concentration during acid fermentation. Notably, Terrisporobacter exhibited a superior acid-producing capability compared to Bacteroides during the dark fermentation of enzymatic hydrolysates. This study provides a new perspective for the practical application of corn stover.

Concentration and crystallization of Microbial Xylitol from oil palm empty fruit bunch (OPEFB) using submerged direct contact membrane distillation (DCMD)

Xylitol possess high solubility in water and increase viscosity at high temperature, making the concentration process become challenging. This study demonstrated the applicability of Membrane Distillation (MD) in concentrating xylitol solutions and evaluates its performance at different conditions and feed compositions. MD was capable of concentrating pure xylitol solution to reach the supersaturated condition. However, when Oil Palm Empty Fruit Bunch (OPEFB) hydrolysate fermentation broth was used, fouling was observed due to the presence of impurities, despite the application of feed agitation. The batch crystallization of xylitol were conducted at 5 °C with a seed addition of 0.5–0.8%-wt. Higher seed concentrations were required to induce crystallization in OPEFB hydrolysate due to lower xylitol concentrations and impurity presence. Furthermore, impurities influenced the quality of the crystals, resulting in crystal purities of 98.98% and 94.56% in the synthetic solution and OPEFB hydrolysate, respectively. These findings indicated notable impact of crystallization temperature, seed addition, and impurity on xylitol crystallization.

Lactic acid production by fermentation of hydrolysate of the macroalga Gracilaria corticata by Lactobacillus acidophilus

Macroalgae (Ulva fasciata, Gracilaria corticata, and Sargassum wightii) were collected from the marine environment and used as the substrate for lactic acid production. These macroalgae were pretreated with hydrochloric acid (0.2 to 0.4 N) for various times (20 to 60 min). Additionally, the algal hydrolysate was incubated with cellulase for 24 h at 30 ± 1 °C to achieve enzymatic saccharification. Proximate analysis of these macroalgae was performed, and the yield was high in G. corticata. The G. corticata hydrolysate was composed of 10.01 ± 0.12% ash content, 1.25 ± 0.2% total fat, 10.2 ± 0.1% crude protein, 9.2 ± 0.2% moisture content, and a higher level of total carbohydrate (69.33 ± 1.5%) than the other two macroalgae. In G. corticata, the enzymatic treatment showed the maximum reducing sugar (33.5 ± 2.3%) relative to the other macroalgal hydrolysates and was considered for optimization of lactic acid production. Lactobacillus acidophilus (MTCC447) utilized pretreated G. corticata hydrolysate (enriched with 5% yeast extract), and maximum lactic acid yield was achieved after 72 h, 30 °C incubation temperature, and 6% inoculum (1×108 CFU/mL) in static culture condition. Batch fermentation was performed in the 1-L bioreactor at room temperature (30 °C) for 96 h. Lactic acid production was maximum within 72 h and the pH value was depleted. The present finding indicates that G. corticata could be used as a substrate for lactic acid production.

Separation of 2,3-Butanediol from Fermentation Broth via Cyclic and Simulated Moving Bed Adsorption Over Nano-MFI Zeolites.

The biomass-based platform molecule 2,3-butanediol (2,3-BDO) has a wide range of applications in production of sustainable fuels, chemicals, synthetic rubber, and others. However, the selective separation of 2,3-BDO from multicomponent fermentation broths presents challenges due to its low concentration, high solubility in water, high boiling point, and presence of many other species. Here, we demonstrate remarkably selective enrichment and recovery of 2,3-BDO from a corn stover hydrolysate fermentation broth by a pure-silica nano-MFI-type zeolite adsorbent. By means of cyclic and simulated moving bed adsorption processes, we obtained concentrated aqueous 2,3-BDO streams from the fermentation process stream with ∼93% purity and 3-fold enrichment, and >98% purity and 8-fold enrichment, respectively. These findings provide strong support for large-scale adsorptive separation for biobased 2,3-BDO production.

S. cerevisiae ERG5DeltaNTH1DeltaAMS1Delta Construction Enhancing Stress Tolerance for Ethanol Production Increase in the Presence of Inhibitors and Mechanism Analysis based on the Comparative Transcriptomics

Saccharomyces cerevisiae is considered the most promising large-scale production strain with ethanol as the main product. The fermentation of Saccharomyces cerevisiae is generally inhibited under various stress conditions. Various inhibitors in the hydrolysate severely inhibit yeast proliferation and yeast accumulation. In this study, S. cerevisiae ERG5, NTH1, and AMS1 were knocked out to improve the yeast stress tolerance by the Clustered Regularly Interspaced Short Palindromic Repeats Cas9 (CRISPR-Cas9) technology. The result indicated that the stress tolerance of S. cerevisiae ERG5ΔNTH1ΔAMS1Δ mutant (S. cerevisiae SCENA) was remarkably improved compared with the wild-type strain. The contents of fecosterol, trehalose, and mannan in S. cerevisiae SCENA were 1.67, 1.53, and 1.47 folds compared with those in the control. The ethanol concentration in S. cerevisiae SCENA reached 16.5 g/L, which was 1.23 folds compared with the control using rice bran hydrolysate. Further, the transcriptome analysis indicated down-regulated differential expression genes (DEGs) in S. cerevisiae SCENA were mainly from cellular response to glucose, cell periphery, and plasma membrane. Up-regulated DEGs were mainly from spore wall assembly, fungal-type cell wall assembly, and ascospore wall assembly. Thus, S. cerevisiae SCENA could effectively produce ethanol using the fermentation of lignocellulosic hydrolysate in the presence of inhibitors by regulating fecosterol, trehalose, and mannan metabolisms.

Brewery spent grain valorization through fermentation: Targeting biohydrogen, carboxylic acids and methane production

This study investigated three different fermentation approaches to explore the potential for producing biohydrogen, carboxylic acids, and methane from hydrolysates of thermally dilute acid pretreated brewer's spent grains (BSG). Initially, the research focused on maximizing the volumetric hydrogen production rate (HPR) in the continuous dark fermentation (DF) of BSG hydrolysates by varying the hydraulic retention time (HRT). The highest HPR reported to date of 5.9 NL/L-d was achieved at 6 h HRT, with a Clostridium-dominated microbial community. The effect of the operational pH (4, 5, 6, and 7) on the continuous acidogenic fermentation was then investigated. A peak carboxylic acid concentration of 17.3 g CODequiv./L was recorded at pH 6, with an associated volumetric productivity of 900.5 ± 13.1 mg CODequiv./L-h and a degree of acidification of 68.3 %. Lactic acid bacteria such as Limosilactobacillus and Lactobacillus were dominant at pH 4–5, while Weissella, Enterococcus, and Lachnoclostridium appeared at pH 6 and 7. Finally, this study evaluated the biochemical methane potential of the DF broth and the unfermented hydrolysates and found high methane yields of 659 and 517 NmL CH4/g-VSadded, respectively, both within one week. Overall, the results showed that pretreated BSG can be a low-cost feedstock for the production of bioenergy and valuable bio-based chemicals in a circular economy.

Effect of Corynebacterium glutamicum Fermentation on the Volatile Flavors of the Enzymatic Hydrolysate of Soybean Protein Isolate.

This study focused on improving the flavor quality of seasonings, and enzymatic hydrolysis of soybean protein isolate (SPI) seasoning via traditional technology may lead to undesirable flavors. Herein, we aimed to develop a new type of SPI seasoning through microbial fermentation to improve its flavor quality. The effect of Corynebacterium glutamicum fermentation on the flavoring compounds of seasonings in SPI enzymatic hydrolysate was examined. Sensory evaluation showed that the SPI seasoning had mainly aromatic and roasted flavor, and the response signals of S18 (aromatic compounds), S24 (alcohols and aldehydes), and S25 (esters and ketones) sensors of the electronic nose differed significantly. Overall, 91 volatile compounds were identified via gas chromatography-mass spectrometry. SPI seasonings contained a higher number of alcohols, ketones, aromatics, and heterocyclic compounds than traditional seasonings, which had stronger cheese, fatty, and roasted aromas. According to the relative odor activity value (ROAV) analysis, n-pentylpyrzine, 2,6-dimethylpyrazine, and tetramethylpyrazine are the key flavoring compounds (ROAV ≥ 1) of SPI seasoning, which may impart a unique roasted and meaty aroma. Therefore, the fermentation of SPI enzymatic hydrolysate with C. glutamicum may improve the flavor quality of its products, providing a new method for the development and production of new seasoning products.

Toxicants improve glycerol production in the fermentation of undetoxified hydrolysate by Candida glycerinogenes.

Toxicants inhibit microbial fermentation and reduce product titres. This work investigated the glycerol production characteristics of Candida glycerinogenes in highly toxic unwashed undetoxified hydrolysate and provided new ideas for high glycerol production from hydrolysates. The unwashed hydrolysate contains higher concentrations of toxicants, such as furfural, acetic acid, phenols and NaCl than the washed alkali-treated bagasse hydrolysate. C. glycerinogenes fermented unwashed undetoxified hydrolysate yielded 36.1g/L glycerol, 15.8% higher than the washed hydrolysate, suggesting that the toxicants stimulated glycerol synthesis. qRT-PCR analysis showed that toxicants of unwashed undetoxified hydrolysates greatly up-regulated the transcript levels of the genes GPD1, HXT4 and MSN4 et al. Overexpressing the above genes increased glycerol production by 27.9% to 46.1g/L. And it was further increased by 8.8% to 50.1g/L in a 5L bioreactor. This result proves that toxicants in lignocellulosic hydrolysates can increase the titre of microbial glycerol production.

Separate hydrolysis and fermentation of softwood bark pretreated with 2-naphthol by steam explosion

Background2-Naphthol, a carbocation scavenger, is known to mitigate lignin condensation during the acidic processing of lignocellulosic biomass, which may benefit downstream processing of the resulting materials. Consequently, various raw materials have demonstrated improved enzymatic saccharification yields for substrates pretreated through autohydrolysis and dilute acid hydrolysis in the presence of 2-naphthol. However, 2-naphthol is toxic to ethanol-producing organisms, which may hinder its potential application. Little is known about the implications of 2-naphthol in combination with the pretreatment of softwood bark during continuous steam explosion in an industrially scalable system.ResultsThe 2-naphthol-pretreated softwood bark was examined through spectroscopic techniques and subjected to separate hydrolysis and fermentation along with a reference excluding the scavenger and a detoxified sample washed with ethanol. The extractions of the pretreated materials with water resulted in a lower aromatic content in the extracts and stronger FTIR signals, possibly related to guaiacyl lignin, in the nonextractable residue when 2-naphthol was used during pretreatment. In addition, cyclohexane/acetone (9:1) extraction revealed the presence of pristine 2-naphthol in the extracts and increased aromatic content of the nonextractable residue detectable by NMR for the scavenger-pretreated materials. Whole-slurry enzymatic saccharification at 12% solids loading revealed that elevated saccharification recoveries after 48 h could not be achieved with the help of the scavenger. Glucose concentrations of 16.9 (reference) and 15.8 g/l (2-naphthol) could be obtained after 48 h of hydrolysis. However, increased inhibition during fermentation of the scavenger-pretreated hydrolysate, indicated by yeast cell growth, was slight and could be entirely overcome by the detoxification stage. The ethanol yields from fermentable sugars after 24 h were 0.45 (reference), 0.45 (2-naphthol), and 0.49 g/g (2-naphthol, detoxified).ConclusionThe carbocation scavenger 2-naphthol did not increase the saccharification yield of softwood bark pretreated in an industrially scalable system for continuous steam explosion. On the other hand, it was shown that the scavenger's inhibitory effects on fermenting microorganisms can be overcome by controlling the pretreatment conditions to avoid cross-inhibition or detoxifying the substrates through ethanol washing. This study underlines the need to jointly optimize all the main processing steps.

Determination of Reducing Sugar Groups from Hydrolysis of Arthrospira platensis Microalgae using Microwaves

The depletion of fossil fuel sources and increasing carbon dioxide (CO2) emissions have encouraged research into renewable energy sources. Bioethanol is an environmentally friendly energy source with considerable potential for reducing dependence on gasoline. Bioethanol is produced from the fermentation process of monosaccharides. The first and second generations of bioethanol are derived from food crops, agricultural waste, and plantation waste, whereas the third generation is produced from microalgae. Arthrospira platensis is a carbohydrate-rich microalgae. This study attempts to determine the content of reducing sugar groups which are monosaccharides formed from a hydrolysis with microwaves. A total of 10 g of microalgae powder was added to 100 mL of 0.3 M H2SO4 solution. The hydrolysis process was carried out in a microwave reactor at a temperature of 100°C for 90 minutes. The hydrolysate obtained was then fermented with Saccharomyces cerevisiae anaerobically in a shaking water bath. The High Performance Liquid Chromatography (HPLC) test was performed to identify reducing sugar groups in the hydrolysate, and the Gas Chromatography (GC) test was performed to determine the concentration of bioethanol produced during the fermentation process. Meanwhile, the solid content of biochar that remained after hydrolysis was analyzed using Fourier-Transform Infrared Spectoscopy (FTIR). The HPLC test findings showed that the glucose concentrations before and after fermentation were 10.52 and 1.91 g/L, respectively, indicating that 81.8% of the glucose was converted to bioethanol. Furthermore, the distillation results from the fermented hydrolysate were analyzed using GC, yielding a bioethanol content of 3.90 g/L.

Enhanced biobutanol production with sustainable Co-substrates synergy from paper waste and garden waste with municipal biowaste

Synergy in the co-processing of lignocellulosic wastes and municipal biowaste (MB) can unlock their potential for biobutanol production. This study assessed the potential for biobutanol production through the co-processing of lignocellulosic waste and MB. Specifically, it compared the co-processing of paper waste with MB to that of garden waste and MB. Ethanol organosolv pretreatment served as a dual-function process for both pretreatment and detoxification purposes. Initial fermentation of hydrolysates from untreated paper waste using Clostridium acetobutylicum produced 0.9 g/L of acetone and ethanol but no detectable butanol. Organosolv pretreatment led to a significant increase in acetone and ethanol production but did not yield butanol. Co-processing paper waste with MB using organosolv pretreatment resulted in the production of 2.8–3.2 g/L butanol, along with increased acetone and ethanol production. Furthermore, co-processing a 1:1 (w/w) mixture of paper waste and MB under mild and severe pretreatment conditions produced 45.5 g and 43.4 g butanol, respectively, compared to 34.8 g and 14.4 g butanol when processing these waste streams separately. The study also explored the positive impact of co-processing garden waste with MB, a distinct lignocellulosic source, enhancing acetone-butanol-ethanol (ABE) yield by 27–40%. These findings highlight the potential of synergistic waste co-processing for achieving a more suitable balance of nutrients to enhance biobutanol and ABE production from biowastes. Additionally, the simultaneous treatment of lignocellulosic waste and municipal biowaste offers a simplified approach to waste processing, contributing to advancements in sustainable biomass utilization and bioenergy production.

The Macroalga Kappaphycus alvarezii as a Potential Raw Material for Fermentation Processes within the Biorefinery Concept: Challenges and Perspectives

Seaweed is a fast-growing biomass source that is currently studied as feedstock for sustainable industrial production in a wide variety of markets. Being composed mostly of polysaccharides, macroalgae can be integrated in biorefineries for obtaining bioproducts via fermentation. Kappaphycus alvarezii has been introduced experimentally to Brazil’s south coastline in 1995 and is now cultivated on a large scale to keep up with the high carrageenan demand in various industrial sectors. In this review article, an introduction is given on renewable biomass and environmental issues, focusing especially on third-generation biomass and its promising features and use advantages. Later on, the processing of K. alvarezii for the use of its saccharide portion for fermentative processes is approached. The current state of research conducted alongside challenges and hurdles in K. alvarezii hydrolysate fermentation processes provides insight into future studies needed to make new fermentation processes viable. Next, some fermentation products are discussed, and the metabolism of galactose in microorganisms is also presented to bring to light other possible fermentation products that are not yet, but can be, obtained from K. alvarezii. Finally, a simple and comprehensive scheme for K. alvarezii fermentation biorefinery is presented to demonstrate a generic example for a possible configuration for obtaining valuable bio-products. In the literature, production of ethanol and lactic acid were already reported from K. alvarezii. This review aims to help envision new industrial processes that can be developed for this most valuable macroalga.

Sustainable wheat straw pretreatment process by self-produced and cyclical crude lactic acid

The purpose of this study was to investigate an environmentally friendly and recyclable pretreatment approach that would enhance the enzymatic digestibility of wheat straw. Wheat straw was pretreated using self-produced crude lactic acid obtained from enzymatic hydrolysate fermentation by Bacillus coagulans. Experimentally, crude lactic acid at low concentration could achieve a pretreatment effect comparable to that of commercial lactic acid. After pretreatment at 180 °C for 60 min with 2.0 % crude lactic acid, hemicellulose could be effectively separated and high recovery of cellulose was ensured, achieving cellulose recovery rate of 95.5 % and hemicellulose removal rate of 92.7 %. Excellent enzymatic hydrolysis was accomplished with a glucose yield of 99.7 %. Moreover, the crude lactic acid demonstrated acceptable pretreatment and enzymatic hydrolysis performance even after three repeated cycles. This not only effectively utilizes the pretreatment solution, but also offers insights into biomass pretreatment using other fermentable acids.

Saccharomyces cerevisiae strains performing similarly during fermentation of lignocellulosic hydrolysates show pronounced differences in transcriptional stress responses.

The need for sustainable alternatives to oil-based production of biochemicals and biofuels is undisputable. Saccharomyces cerevisiae is the most commonly used industrial fermentation workhorse. The fermentation of lignocellulosic hydrolysates, second-generation biomass unsuited for food and feed, is still hampered by lowered productivities as the raw material is inhibitory for the cells. In order to map the genetic responses of different S. cerevisiae strains, we performed RNA sequencing of a CEN.PK laboratory strain, two industrial strains (KE6-12 and Ethanol Red), and two wild-type isolates of the LBCM collection when cultivated anaerobically in wheat straw hydrolysate. While the response to inhibitors of S. cerevisiae has been studied earlier, this has in previous studies been done in aerobic conditions. The transcriptomic analysis highlights different evolutionary adaptations among the different S. cerevisiae strains and suggests novel gene targets for improving fermentation performance and robustness.

Black soldier fly bioconversion to cultivated meat media components using blue catfish gut microbiome

This study aimed to develop low-cost media for cell-cultivated seafood using gut-microbial community-assisted fermentation. Black soldier fly larvae (Hermetia illucens) were used as the substrate and exposed to gut microbial communities isolated from blue catfish (Ictalurus furcatus). Fermentation led to a decrease in species richness, with the loss of important chitinase and protease-producing genera such as Pseudomonas and Clostridiaceae. However, there was an increase in Paraclostridium and members of the Enterobacteriaceae. In addition, the effect of fermented hydrolysates from BSFL on the proliferation of zebrafish embryo fibroblasts was tested in comparison to fetal bovine serum (FBS) in in vitro cell cultivation. Lower concentrations of FBS resulted in decreased cell density and altered cell morphology. The supplementation of hydrolysate B at high peptide concentrations had cytotoxic effects on the cells, while at lower peptide concentrations, it improved cell proliferation only in cultures with 2.5 % FBS.

Green Macroalgae Hydrolysate for Biofuel Production: Potential of Ulva rigida

In this study, the green macroalgae Ulva rigida, which contains 34.9% carbohydrates, underwent treatment with commercial hydrolytic enzymes. This treatment yielded a hydrolysate that contained 23 ± 0.6 g·L−1 of glucose, which was subsequently fermented with Saccharomyces cerevisiae. The fermentation process resulted in an ethanol concentration of 9.55 ± 0.20 g·L−1. The optimal conditions for ethanol production by S. cerevisiae were identified as follows: non-sterilized conditions, an absence of enrichment, and using an inoculum size of 118 mg·L−1. Under these conditions, the fermentation of the green macroalgal hydrolysate achieved a remarkable conversion efficiency of 80.78%. The ethanol o/t ratio, namely the ratios of the experimental to theoretical ethanol produced, for Scheffersomyces stipitis, Candida guilliermondii, Kluyveromyces marxianus, and S. cerevisiae after 48 h of fermentation were 52.25, 63.20, 70.49, and 82.87%, respectively. Furthermore, S. cerevisiae exhibited the best outcomes in terms of ethanol production (9.35 g·L−1) and conversion efficiency (80.78%) after 24 h (optimal time) of fermentation.

Production of Medium Chain Length Polyhydroxyalkanoate from Waste Cannabis sativa Biomass

This works aims to evaluate a process to convert residual medical Cannabis sativa stalks into medium chain length polyhydroxyalkanoate (mcl-PHA), using chemical pretreatment and enzymatic hydrolysis to obtain sugars as a carbon source for a fermentation process with Pseudomonas aeruginosa. Chemical composition analysis revealed a content of structural polysaccharides of 57.64%. Thermochemical pretreatments with 2% sulfuric acid or 2% sodium hydroxide were capable of partially removing hemicellulose and lignin, as well as increasing cellulose crystallinity. Pretreated biomass was subjected to hydrolysis using commercial cellulase cocktails Celluclast® 1.5 L and Cellic® CTec3. Acid pretreatment showed an adverse effect on hydrolysis yield of holocellulose, decreasing to 39.5%; compared to 44.4% of untreated biomass. Alkaline pretreatment increased degree of hydrolysis up to 73.3%. Shake flask fermentation of hydrolysate with Pseudomonas aeruginosa produced cell growth of 1.65 g/L and a mcl-PHA titer of 0.41 g/L. Extracted polymer presented characteristic FTIR bands for PHAs, glass transition temperature of − 50.8 °C, melting temperature of 48.9 °C, possibly allowing its use in the biomedical industry. The developed process represents a potential way to valorize Cannabis waste stalks, using alkaline pretreatment, enzymatic hydrolysis with Celluclast® 1.5 L and fermentation with Pseudomonas aeruginosa. Further work should focus on improving yield of the obtained polyhydroxyalkanoate in fermentation, in order to improve industrial feasibility of the entire process and, in turn, increasing revenue of the medical Cannabis industry.Graphical

Biorefinery design with combined deacetylation and microwave pretreatment for enhanced production of polyhydroxyalkanoates and efficient carbon utilization from lignocellulose

Lignocellulosic biomass has emerged as a sustainable and economically viable feedstock for polyhydroxyalkanoate (PHA) production. However, it has seldom been used as part of an integrated process for effective carbon utilization. An innovative combined deacetylation and microwave pretreatment strategy (DEA-MW) and a wild-type Cupriavidus necator strain with the potential to utilize a lignocellulose-based carbon source and PHA biosynthesis were selected to set up the module needed to enable a new sustainable platform for biomass pretreatment and waste carbon valorization. DEA-MW pretreatment was introduced to mitigate the concentration of inhibitory acetic acid and phenolic compounds in the biomass hydrolysate. This pretreatment enhanced deacetylation and delignification, resulting in 95.50 % deacetylation and 45.30 % delignification. When DEA-MW pretreated rice husk biomass was coupled with microbial fermentation, C. necator promoted the fermentability of the rice husk hydrolysate. With a 3 g/L of levulinic acid from hydrolysis, as the sole carbon source, we achieved a concentration of PHA 440 mg/L with a content of 65 %. We also successfully recovered 72.22 % of lignin using a combined DEA-MW pretreatment. A detailed structural analysis showed that β-O-4, β–β, and β-5 linkages had been preserved in the recovered lignin. This study has shown that DEA-MW pretreatment plays a role in promoting biomass conversion and enhancing PHA production and opens up new possibilities for efficient and cost-effective lignocellulose-based biorefinery processes.