Characterization of sugarcane bagasse solubilization and utilization by thermophilic cellulolytic and saccharolytic bacteria at increasing solid loadings.

In Brazil the main feedstock used for ethanol production is sugarcane juice, resulting in large amounts of bagasse. Bagasse has high potential for cellulosic ethanol production, and consolidated bioprocessing (CBP) has potential for lowering costs. However, economic feasibility requires bioprocessing at high solids loadings, entailing engineering and biological challenges. This study aims to document and characterize carbohydrate solubilization and utilization by defined cocultures of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum at increasing loadings of sugarcane bagasse. Results show that fractional carbohydrate solubilization decreases as solids loading increases from 10 g/L to 80 g/L. Cocultures enhance solubilization and carbohydrate utilization compared to monocultures, irrespective of initial solids loading. Rinsing bagasse before fermentation slightly decreases solubilization. Experiments studying inhibitory effects using spent media and dilution of broth show that negative effects are temporary or reversible. These findings highlight the potential of converting sugarcane bagasse via CBP, pointing out performance limitations that must be addressed.

Enhanced thermophilic high-solids anaerobic digestion of organic fraction of municipal solid waste with spatial separation from conductive materials in a single reactor volume.

Despite benefits such as lower water and working volume requirements, thermophilic high solids anaerobic digestion (THSAD) often fails due to the rapid build-up of volatile fatty acids (VFAs) and the associated drop in pH. Use of conductive materials (CM) can promote THSAD through stimulation of direct interspecies electron transfer (DIET), while the need for their constant dosing due to poor separation from effluent impairs economic feasibility. This study used an approach of spatially separating magnetite and granular activated carbon (GAC) from the organic fraction of municipal solid waste (OFMSW) in a single reactor for THSAD. GAC and magnetite addition could both mitigate the severe inhibition of methanogenesis after VFAs build-up to ∼28-30 g/L, while negligible methane production was observed in the control group. The highest methane yield (286 mL CH4/g volatile solids (VS)) was achieved in magnetite-added reactors, while the highest maximum CH4 production rates (26.38 mL CH4/g VS/d) and lowest lag-phase (2.83 days) were obtained in GAC-added reactors. The enrichment of GAC and magnetite biofilms with various syntrophic and potentially electroactive microbial groups (Ruminiclostridium 1, Clostridia MBA03, Defluviitoga, Lentimicrobiaceae) in different relative abundances indicates the existence of specific preferences of these groups for the nature of CM. According to predicted basic metabolic functions, CM can enhance cellular processes and signals, lipid transport and metabolism, and methane metabolism, resulting in improved methane production. Rearrangement of metabolic pathways, formation of pili-like structures, enrichment of biofilms with electroactive groups and a significant improvement in THSAD performance was attributed to the enhancement of the DIET pathway. Promising results obtained in this work due to the spatial separation of the bulk OFMSW and CM can be useful for modeling larger-scale THSAD systems with better recovery of CM and cost-effectiveness.

From grass to gas and beyond: Anaerobic digestion as a key enabling technology for a residual grass biorefinery.

Roadside grass clippings hold potential as a sustainable source of bioenergy as they do not compete with crops for land use, and are only partially utilized for low-value applications. In this study, we proposed using roadside grass as a sole feedstock for anaerobic digestion (AD) in three different settings, and assessed the potential of producing biomaterials and fertilizers from grass-based digestate. Wet continuous digestion at pilot scale and dry batch digestion at pilot and large scales resulted in biogas yields up to 700 Nm3.t-1 DOM with a methane content of 49-55 %. Despite promising results, wet AD had operational problems such as clogging and poor mixing; once upscaled, the dry digestion initially also presented an operational problem with acidification, which was overcome by the second trial. Digested grass fibers from the pilot dry AD were processed into biomaterials and performed similarly or better than the undigested fibers, while around 20 % performance reduction was observed when compared to reference wood fibers. A mass balance indicated reduced fiber recovery when higher biogas production was obtained. The liquid fraction from the pilot dry AD was characterized for its nutrient content and used as a biofertilizer in another study. In contrast, the leachate collected from the large-scale dry AD had a low nitrogen content and high chloride content that could hinder its further use. Finally, a regional market analysis was conducted showing that the biocomposites produced with the available grass fibers could substitute at least half of the current European market based on our results.

Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities.

BACKGROUND: The polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear. RESULTS: An LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid). CONCLUSIONS: This study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction.

Enhanced ethanol production from paper sludge waste under high-solids conditions with industrial and cellulase-producing strains of Saccharomyces cerevisiae.

Reported ethanol titres from hydrolysis-fermentation of the degraded fibres in paper sludge (PS) waste, generally obtained under fed-batch submerged conditions, can be improved through fermentation processes at high solids loadings, as demonstrated in the present study with two industrial PS wastes at enzyme dosages appropriate for solids loadings up to 40% (w/w). The industrial yeast,Saccharomyces cerevisiaestrain Ethanol Red®, was compared to two genetically engineeredS. cerevisiaestrains, namely Cellusec® 1.0 and Cellusec® 2.0, capable of xylose utilisation, and xylose utilisation and cellulase production, respectively. High-solids batch fermentations were conducted in 3 L horizontal rotating reactors and ethanol titres of 100.8 and 73.3 g/L were obtained for virgin pulp and corrugated recycle PS, respectively, at 40% (w/w) solids loading using Ethanol Red®. Xylose utilisation by Cellusec® 1.0 improved ethanol titres by up to 10.3%, while exogenous cellulolytic enzyme requirements were reduced by up to 50% using cellulase-producing Cellusec® 2.0.

Achieving efficient and rapid high-solids enzymatic hydrolysis for producing high titer ethanol with the assistance of di-rhamnolipids.

High-solids enzymatic hydrolysis is the premise of obtaining high concentration ethanol by fermentation. In this study, corn stover was first pretreated with formic acid under mild conditions, and more than 70 % of xylan and lignin were removed within the first hour. 173.0 g/L glucose was achieved from total 30 % solid of the pretreated corn stover via fed-batch mode. Moreover, the glucose concentration rose to 194.5 g/L and the hydrolysis time was significantly reduced by 42.9 % with the addition of di-rhamnolipid. On this basis, 89.1 g/L ethanol was obtained by fermentation, and the presence of di-rhamnolipid had no negative effect on fermentation. The effective conversion of corn stover to high titer ethanol provides support for the conversion of stover to ethanol in industrial production.

Addressing two major limitations in high-solids enzymatic hydrolysis by an ordered polyethylene glycol pre-incubated strategy: Rheological properties and lignin adsorption for enzyme.

High-solids enzymatic hydrolysis for biomass has currently received considerable interest. However, the solid effect during the process limits its economic feasibility. This work presented an ordered polyethylene glycol (PEG) pre-incubated strategy for enhancing the auxiliary effect of PEG in a high-solids enzymatic hydrolysis system. The substrate and enzyme were separately pre-incubated with PEG in this strategy. The ordered PEG pre-incubated strategies yielded a maximum glucose concentration of 166.6 g/L from 32 % (w/v) pretreated corncob with an enzymatic yield of 94.1 % by 72 h hydrolysis. Using this method, PEG not only lessened the lignin adsorption to cellulase but also altered particle rheological characteristics in the high-solids enzymatic hydrolysis system as a viscosity modifier. This study offered a new insight into the mechanism behind the PEG synergistic effect and would make it possible to achieve efficient high-solids loading hydrolysis in the commercial manufacture of cellulosic ethanol.

Optimization of membrane filtration and cleaning strategy in a high solid thermophilic AnMBR treating food waste.

Anaerobic membrane bioreactor is advantageous over traditional processes for food waste treatment, i.e. short retention time, high loading rate, and particulate clean permeate. However, establishing a sustainable membrane filtration is a long-standing challenge because of its high viscosity and solids concentration characteristics. Therefore, this study investigated the changes in the membrane permeability before and after the cleaning during a 130-day thermophilic anaerobic experiment. Results show that the AnMBR system could maintain high stability even under a short HRT of 10 days and OLR of 9.0 kg-COD/(m3·d) with low volatile fatty acid of 50 mg/L. The membrane filtration deteriorates with the concurrence of a sharp increase of viscosity when the volatile solids reached 23 g/L. A critical flux was achieved at 5.5 L/(m2·h) under optimized operation conditions, membrane filtration/relaxing ratio with less than 4:1 at a hydraulic retention time of 15 d. Membrane fouling can be removed by soaking the membrane in NaClO (1 g/L, 15 h) and citric acid (2 g/L, 2 h). Conclusively, this work provides insight to establish the operation strategy for a thermophilic AnMBR treating food waste.

Mass Transfer Enhancement in High-Solids Anaerobic Digestion of Organic Fraction of Municipal Solid Wastes: A Review.

The increasing global population and urbanization have led to a pressing need for effective solutions to manage the organic fraction of municipal solid waste (OFMSW). High-solids anaerobic digestion (HS-AD) has garnered attention as a sustainable technology that offers reduced water demand and energy consumption, and an increased biogas production rate. However, challenges such as rheology complexities and slow mass transfer hinder its widespread application. To address these limitations, this review emphasizes the importance of process optimization and the mass transfer enhancement of HS-AD, and summarizes various strategies for enhancing mass transfer in the field of HS-AD for the OFMSW, including substrate pretreatments, mixing strategies, and the addition of biochar. Additionally, the incorporation of innovative reactor designs, substrate pretreatment, the use of advanced modeling and simulation techniques, and the novel conductive materials need to be investigated in future studies to promote a better coupling between mass transfer and methane production. This review provides support and guidance to promote HS-AD technology as a more viable solution for sustainable waste management and resource recovery.

Synthesis of bifunctional thermal response promoters for improved high-solids enzymatic hydrolysis of corncob residues.

The enzymatic hydrolysis cost of lignocellulose can be reduced by improving enzymatic hydrolysis and recycling cellulase by adding additives. A series of copolymers P(SSS-co-SPE) (PSSPs) were synthesized using sodium p-styrene sulfonate (SSS) and sulfobetaine (SPE) as monomers. PSSP exhibited upper critical solution temperature response. PSSP with high molar ratio of SSS displayed more significant improved hydrolysis performance. When 10.0 g/L PSSP5 was added to the hydrolysis system of corncob residues, and substrate enzymatic digestibility at 72 h (SED@72 h) increased by 1.4 times. PSSP with high molecular weight and moderate molar ratio of SSS, had significant temperature response, enhanced hydrolysis, and recovering cellulase properties. For high-solids hydrolysis of corncob residues, SED@48 h increased by 1.2 times with adding 4.0 g/L of PSSP3. Meanwhile, 50% of cellulase amount was saved at the room temperature. This work provides a new idea for reducing the hydrolysis cost of lignocellulose-based sugar platform technology.

Performance of high solids enzymatic hydrolysis and bioethanol fermentation of food waste under the regulation of saponin.

Bioethanol recovery from food waste through high solids enzymatic hydrolysis (HSEH) and high solids bioethanol fermentation (HSBF) alleviate the energy crisis. However, this cause decreased glucose and bioethanol yields due to the high solids content. In this study, saponin was introduced into food waste HSEH and HSBF systems to enhance the product yields. Under the regulation of saponin, the substrate released >90% of the theoretical reducing sugar. The glucose concentration increased by 137.41 g/L after 24 h of HSEH with 2.0% saponin. The bioethanol titer reached 73.2 g/L (1.0%-saponin). Untargeted metabolomics illustrating that saponin had higher antifungal properties at lower concentrations (0.5%-saponin) that caused a decrease in bioethanol yield. The addition of saponin concentrations of 1.0%∼3.0% promoted HSEH, HSBF, and the metabolism of Saccharomyces cerevisiae; thus, 1.0% was suggested for practical use. This study deepened the understanding of saponin in enhancing HSBF and provides theoretical support for further application.

Enhancing of pretreatment on high solids enzymatic hydrolysis of food waste: Sugar yield, trimming of substrate structure.

The development of high solids enzymatic hydrolysis (HSEH) technology is a promising way to improve the efficiency of bioenergy production from solid waste. Pretreatment methods such as ultrasound (USP), freeze-thaw (FTP), hydrothermal (HTP), and dried (DRD) were carried out to evaluate the effect and mechanism of the pretreatment methods on the HSEH of FW. The reducing sugar of HTP and DRD reached 94.75% and 94.92% of the theoretical value. HTP and DRD could reduce the crystallinity of FW. DRD resulted in lower alignment and the occurrence of fractures of the substrate and exposed the α-1,4 glycosidic bond of starch. The high destructive power of HTP and DRD reduced the obstacles caused by the high solid content. Moreover, DRD consumed only 27.62% of the total energy of HTP. DRD could be a promising pretreatment methods for glucose recovery for its high product yield, significant substrate destruction, and economic feasibility.

Inhibitory mechanisms on dry anaerobic digestion: Ammonia, hydrogen and propionic acid relationship.

Inhibitory pathways in dry anaerobic digestion are still understudied and current knowledge on wet processes cannot be easily transferred. This study forced instability in pilot-scale digesters by operating at short retention times (40 and 33 days) in order to understand inhibition pathways over long term operation (145 days). The first sign of inhibition at elevated total ammonia concentrations (8 g/l) was a headspace hydrogen level over the thermodynamic limit for propionic degradation, causing propionic accumulation. The combined inhibitory effect of propionic and ammonia accumulation resulted in further increased hydrogen partial pressures and n-butyric accumulation. The relative abundance of Methanosarcina increased while that of Methanoculleus decreased as digestion deteriorated. It was hypothesized that high ammonia, total solids and organic loading rate inhibited syntrophic acetate oxidisers, increasing their doubling time and resulting in its wash out, which in turn inhibited hydrogenotrophic methanogenesis and shifted the predominant methanogenic pathway towards acetoclastic methanogenesis at free ammonia over 1.5 g/l. C/N increases to 25 and 29 reduced inhibitors accumulation but did not avoid inhibition or the washout of syntrophic acetate oxidising bacteria.

Use of nanobubble water bioaugmented anaerobically digested sludge for high-efficacy energy production from high-solids anaerobic digestion of corn straw.

An increasing attention has been paid to the secure and sustainable management of agricultural wastes, especially lignocellulosic biomass. Nanobubble water (NBW) contains 106-108 bubbles/mL with diameter <1000 nm. Although previous studies have examined the enhancement effects of NBW on methane production from organic solid wastes, the NBW-based anaerobic digestion (AD) system is still restrained from practical application due to the large increase in AD reactor volume, generation of wastewater, and increase in energy consumption as well. In this study, NBW bioaugmentation of anaerobically digested sludge for the first time was performed for high-solids AD of corn straw. Results show that cellulase, xylanases and lignin peroxidase activities were increased by 2-55% during the NBW bioaugmentation process. Significant enrichment of hydrolytic/acidogenic bacteria and methanogenic archaea were noticed in the NBW bioaugmented sludge. This study clearly demonstrated 47% increase in methane production from high-solids AD of corn straw when O2-NBW bioaugmented sludge was applied, achieving a net energy gain of 5138 MJ/t-volatile solids of corn straw with an energy recovery of 34%. The NBW-based high-solids AD system can provide a novel and sustainable management solution for renewable energy production from agricultural wastes, targeting the reduction of environmental pollution and energy crisis.

High solids loading pretreatment: The core of lignocellulose biorefinery as an industrial technology - An overview.

Pretreatment is the first and most determinative, yet the least mature step of lignocellulose biorefinery chain. The current stagnation of biorefinery commercialization indicates the barriers of the existing pretreatment technologies are needed to be unlocked. This review focused on one of the core factors, the high lignocellulose solids loading in pretreatment. The high solids loading of pretreatment significantly reduces water input, energy requirement, toxic compound discharge, solid/liquid separation costs, and carbon dioxide emissions, improves the titers of sugars and biproducts to meet the industrial requirements. Meanwhile, lignocellulose feedstock after high solids loading pretreatment is compatible with the existing logistics system for densification, packaging, storage, and transportation. Both the technical-economic analysis and the cellulosic ethanol conversion performance suggest that the solids loading in the pretreatment step need to be further elevated towards an industrial technology and the effective solutions should be proposed to the technical barriers in high solids loading pretreatment operations.

Prospects of thermotolerant Kluyveromyces marxianus for high solids ethanol fermentation of lignocellulosic biomass.

Simultaneous saccharification and fermentation (SSF) is effective for minimizing sugar inhibition during high solids fermentation of biomass solids to ethanol. However, fungal enzymes used during SSF are optimal between 50 and 60 °C, whereas most fermentative yeast, such as Saccharomyces cerevisiae, do not tolerate temperatures above 37 °C. Kluyveromyces marxianus variant CBS 6556 is a thermotolerant eukaryote that thrives at 43 °C, thus potentially serving as a promising new host for SSF operation in biorefineries. Here, we attempt to leverage the thermotolerance of the strain to demonstrate the application of CBS 6556 in a high solids (up to 20 wt% insoluble solid loading) SSF configuration to understand its capabilities and limitations as compared to a proven SSF strain, S. cerevisiae D5A. For this study, we first pretreated hardwood poplar chips using Co-Solvent Enhanced Lignocellulosic Fractionation (CELF) to remove lignin and hemicellulose and to produce cellulose-enriched pretreated solids for SSF. Our results demonstrate that although CBS 6556 could not directly outperform D5A, it demonstrated similar tolerance to high gravity sugar solutions, superior growth rates at higher temperatures and higher early stage ethanol productivity. We discovered that CBS 6556's membrane was particularly sensitive to higher ethanol concentrations causing it to suffer earlier fermentation arrest than D5A. Cross-examination of metabolite data between CBS 6556 and D5A and cell surface imaging suggests that the combined stresses of high ethanol concentrations and temperature to CBS 6556's cell membrane was a primary factor limiting its ethanol productivity. Hence, we believe K. marxianus to be an excellent host for future genetic engineering efforts to improve membrane robustness especially at high temperatures in order to achieve higher ethanol productivity and titers, serving as a viable alternative to D5A.

Subcritical Water as Pretreatment Technique for Bioethanol Production from Brewer's Spent Grain within a Biorefinery Concept.

Bioeconomy and environmental issues envisage industrial by-products such as Brewer's spent grain (BSG) as renewable resources for their recycling and reuse within a biorefinery concept. This study aimed to investigate the production of bioethanol from subcritical water (subW) pretreated BSG, following the conversion of the BSG biopolymers cellulose and hemicelluloses. The subW pretreatment was performed in a batch reactor at 174 °C, during 60 min and 5% (w/v) of dry BSG charge. The behavior of BSG biopolymers under subW pretreatment was monitored by evaluating the chemical composition of the liquid and solid streams and the chemical and structural changes caused in the solid residues by scanning electron microscope (SEM), CHNS elemental analysis and water retention value (WRV). The production of bioethanol from subW-pretreated BSG was assessed by separate hydrolysis and fermentation (SHF) and also by simultaneous saccharification and fermentation (SSF) by using the enzymatic cocktail Celluclast 1.5 L (40 FPU/gsolids) and the yeast Ethanol Red®. The higher bioethanol productivity (1.073 g∙L-1∙h-1) and concentration (32.18 g/L) were achieved by SSF with higher solids' loading (25%) and following a fed-batch strategy. These results suggest that subcritical water pretreatment is a promising technology for the valorization of BSG as a feedstock for second-generation bioethanol production.

Bioethanol production from microalgae biomass at high-solids loadings.

Industrial adoption of microalgae biofuel technology has always been hindered by its economic viability. To increase the feasibility of bioethanol production from microalgae, fermentation was applied to Chlorella vulgaris FSP-E biomass at high-solids loading conditions. First, Chlorella vulgaris FSP-E was cultivated to produce microalgae biomass with high carbohydrate content. Next, different ethanol-producing microorganisms were screened. Saccharomyces cerevisiae FAY-1 showed no inhibition when fermenting high initial glucose concentrations and was selected for the fermentation experiments at high-solids loadings. Optimization of acid hydrolysis at high biomass loading was also performed. The fermentation of microalgal biomass hydrolysate produced a final ethanol concentration and yield higher than most reported literature using microalgae feedstock. In addition, the kinetics of bioethanol fermentation of microalgae hydrolysate under high-solids loading were evaluated. These results showed the potential of fermenting microalgae biomass at high-solids loading in improving the viability of microalgae bioethanol production.

Anaerobic Digestion of Pig-Manure Solids at Low Temperatures: Start-Up Strategies and Effects of Mode of Operation, Adapted Inoculum, and Bedding Material.

The objective of this study was to obtain start-up strategies for the operation of a dry anaerobic digestion (DAD) system treating pig-manure (PM) solids at low-temperatures, and evaluate the effects of operation mode, adapted inoculum, and bedding material on the performance. A DAD system coupled with an inoculum system (two-stage DAD) was operated at 20 ± 1 °C to digest PM solids (Total Solids, TS: 27%) with wheat straw or woodchips as bedding materials (TS substrate-mixture: 45%) using a liquid inoculum. Static DAD was also operated in parallel for comparison purposes. Overall, the percolation-recirculation mode of operation was superior to the static mode; the former had more than a 3-fold increase in specific methane yield in cycle 3. Using the adapted inoculum in cycle-2 improved methane yield by 7% and 26% for cycles 1 and 3, respectively, under the percolation-recirculation mode of operation. In addition, the digestate resulting from the digestion of woodchips + PM solids had better physical characteristics than wheat straw + PM solids. Thus, anaerobic digestion of pig-manure solids at low-temperatures with appropriate start-up strategies, inoculum, and bedding material is a promising technology for transforming PM solids into biogas and using its digestate as biofertilizer.

Carbon cloth amendment for boosting high-solids anaerobic digestion with percolate recirculation: Spatial patterns of microbial communities.

The addition of conductive materials in anaerobic digestion (AD) is a promising method for boosting biomethane recovery from organic waste. However, conductive additives have rarely been investigated for the high-solids anaerobic digestion (HSAD). Here, the impact of adding carbon cloth in the solid phase of an HSAD system with percolate recirculation was investigated. Furthermore, spatial patterns of microbial communities in suspended biomass, percolate, and carbon cloth attached biofilm were assessed. Carbon cloth increased biomethane yield from source-separated organics (SSO) by 20% more than the unamended control by shortening the lag phase (by 15%) and marginally improving the methanogenesis rate constant (by ∼8%) under a batch operation for 50 days. Microbial community analysis demonstrated higher relative abundances of the archaeal population in the carbon cloth amended reactor than in unamended control (12%-21% vs. 5%-15%). Compared to percolate and suspension, carbon cloth attached microbial community showed higher enrichment of known electroactive Pseudomonas species along with Methanosarcina and Methanobacterium species, indicating the possibility of DIET-based syntrophy among these species.