Perspective on Lignin Conversion Strategies That Enable Next Generation Biorefineries.

The valorization of lignin, a currently underutilized component of lignocellulosic biomass, has attracted attention to promote a stable and circular bioeconomy. Successful approaches including thermochemical, biological, and catalytic lignin depolymerization have been demonstrated, enabling opportunities for lignino-refineries and lignocellulosic biorefineries. Although significant progress in lignin valorization has been made, this review describes unexplored opportunities in chemical and biological routes for lignin depolymerization and thereby contributes to economically and environmentally sustainable lignin-utilizing biorefineries. This review also highlights the integration of chemical and biological lignin depolymerization and identifies research gaps while also recommending future directions for scaling processes to establish a lignino-chemical industry.

Simulating Rumen Conditions Using an Anaerobic Dynamic Membrane Bioreactor to Enhance Hydrolysis of Lignocellulosic Biomass.

An anaerobic dynamic membrane bioreactor (AnDMBR) mimicking rumen conditions was developed to enhance the hydrolysis of lignocellulosic materials and the production of volatile fatty acids (VFAs) when treating food waste. The AnDMBR was inoculated with cow rumen content and operated at a 0.5 day hydraulic retention time, 2-4 day solids retention time, a temperature of 39 °C, and a pH of 6.3, characteristics similar to those of a rumen. Removal rates of neutral detergent fiber and acid detergent fiber of 58.9 ± 8.4 and 69.0 ± 8.6%, respectively, and a VFA yield of 0.55 ± 0.12 g VFA as chemical oxygen demand g volatile solids (VS)fed-1 were observed at an organic loading rate of 18 ± 2 kg VS m-3 day-1. The composition and activity of the microbial community remained consistent after biofilm disruption, bioreactor upset, and reinoculation. Up to 66.7 ± 5.7% of the active microbial populations and 51.0 ± 7.0% of the total microbial populations present in the rumen-mimicking AnDMBR originated from the inoculum. This study offers a strategy to leverage the features of a rumen; the AnDMBR achieved high hydrolysis and fermentation rates even when treating substrates different from those fed to ruminants.

Simultaneous carbon catabolite repression governs sugar and aromatic co-utilization in Pseudomonas putida M2.

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (p-coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p-coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+p-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).IMPORTANCEA newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

Evaluation of Nanaerobic Digestion as a Mechanism to Explain Surplus Methane Production in Animal Rumina and Engineered Digesters.

Nanaerobes are a newly described class of microorganisms that use a unique cytochrome bd oxidase to achieve nanaerobic respiration at <2 µM dissolved oxygen (∼1% of atmospheric oxygen) but are not viable above this value due to the lack of other terminal oxidases. Although sharing an overlapping ecological niche with methanogenic archaea, the role of nanaerobes in methanogenic systems has not been studied so far. To explore their occurrence and significance, we re-analyzed published meta-omic datasets from animal rumina and waste-to-energy digesters, including conventional anaerobic digesters and anaerobic digesters with ultra-low oxygenation. Results show that animal rumina share broad similarities in the microbial community and system performance with oxygenated digesters, rather than with conventional anaerobic digesters, implying that trace levels of oxygen drive the efficient digestion in ruminants. The rumen system serves as an ideal model for the newly named nanaerobic digestion, as it relies on the synergistic co-occurrence of nanaerobes and methanogens for methane yield enhancement. The most abundant ruminal bacterial family Prevotellaceae contains many nanaerobes, which perform not only anaerobic fermentation but also nanaerobic respiration using cytochrome bd oxidase. These nanaerobes generally accompany hydrogenotrophic methanogens to constitute a thermodynamically and physiologically consistent framework for efficient methane generation. Our findings provide new insights into ruminal methane emissions and strategies to enhance methane generation from biomass.

Competitive Reactions during Ethanol Chain Elongation Were Temporarily Suppressed by Increasing Hydrogen Partial Pressure through Methanogenesis Inhibition.

Organic waste streams can be converted into high-value platform chemicals such as medium-chain carboxylic acids (MCCAs) using mixed microbial communities via chain elongation. However, the heterogeneity of waste streams and the use of complex microbial communities can lead to undesirable reactions, thus decreasing process efficiency. We explored suppressing excessive ethanol oxidation to acetate (EEO) by increasing the hydrogen partial pressure (PH2) through hydrogenotrophic methanogenesis inhibition by periodically adding 2-bromoethanesulfonate (2-BES) to an MCCA-producing bioreactor to reach 10 mM of 2-BES upon addition. The bioreactor was fed with pretreated food waste and brewery waste containing high concentrations of short-chain carboxylic acids and ethanol, respectively. While 2-BES addition initially reduced EEO, some methanogens (Methanobrevibacter spp.) persisted and resistant populations were selected over time. Besides changing the methanogenic community structure, adding 2-BES also changed the bacterial community structure due to its impact on PH2. While we demonstrated that PH2 could be manipulated using 2-BES to control EEO, methods that do not require the addition of a chemical inhibitor should be explored to maintain optimum PH2 for long-term suppression of EEO.

Anaerobic co-digestion: Current status and perspectives.

Anaerobic digestion is a long-established technology for the valorization of diverse organic wastes with concomitant generation of valuable resources. However, mono-digestion (i.e., anaerobic digestion using one feedstock) suffers from challenges associated with feedstock characteristics. Co-digestion using multiple feedstocks provides the potential to overcome these limitations. Significant research and development efforts have highlighted several inherent merits of co-digestion, including enhanced digestibility due to synergistic effects of co-substrates, better process stability, and higher nutrient value of the produced co-digestate. However, studies focused on the underlying effects of diverse co-feedstocks on digester performance and stability have not been synthesized so far. This review fills this gap by highlighting the limitations of mono-digestion and critically examining the benefits of co-digestion. Furthermore, this review discusses synergistic effect of co-substrates, characterization of microbial communities, the prediction of biogas production via different kinetic models, and highlights future research directions for the development of a sustainable biorefinery.

Synergistic association between cytochrome bd-encoded Proteiniphilum and reactive oxygen species (ROS)-scavenging methanogens in microaerobic-anaerobic digestion of lignocellulosic biomass.

Intermittent (every other day) microaerobic [picomolar oxygen by oxidation-reduction potential (ORP) set at +25 mV above anaerobic baseline] digestion of lignocellulosic biomass showed higher digestibility and better stability at a high organic loading rate (OLR) of 5 g volatile solids (VS)/L/d than that under strict anaerobic conditions. However, the microbial mechanisms supporting the delicate balance under microaeration remain underexplored. On the basis of our previous findings that microbial communities in replicate experiments were dominated by strains of the genus Proteiniphilum but contained diverse taxa of methanogenic archaea, here we recovered related genomes and reconstructed the putative metabolic pathways using a genome-centric metagenomic approach. The highly enriched Proteiniphilum strains were identified as efficient cellulolytic facultative bacterium, which directly degraded lignocellulose to carbon dioxide, formate, and acetate via aerobic respiration and anaerobic fermentation, alternatively. Moreover, high oxygen affinity cytochromes, bd-type terminal oxidases, in Proteiniphilum strains were found to be closely associated with such picomolar oxygen conditions, which has long been overlooked in anaerobic digestion. Furthermore, hydrogenotrophic methanogenesis was the prevalent pathway for methane production while Methanosarcina, Methanobrevibacter, and Methanocorpusculum were the dominant methanogens in the replicate experiments. Importantly, the two functional groups, namely cellulolytic facultative Proteiniphilum strains and methanogens, encoded various antioxidant enzymes. Energy-dependent reactive oxygen species (ROS) scavengers (superoxide reductase (SOR) and rubrerythrin (rbr) were ubiquitously present in different methanogenic taxa in response to replicate-specific ORP levels (-470, -450 and -475 mV). Collectively, cytochrome bd oxidase and ROS defenders may play roles in improving the digestibility and stability observed in intermittent microaerobic digestion.

Intermittent micro-aeration: New strategy to control volatile fatty acid accumulation in high organic loading anaerobic digestion.

This study developed an intermittent oxidation-reduction potential (ORP)-controlled micro-aeration system for high solids anaerobic digestion (AD) of lignocellulosic biomass without volatile fatty acids (VFA) accumulation at high organic loading rate (OLR). Traditional AD of Napier grass, a model lignocellulosic biomass, at an OLR of 5 g volatile solids (VS)/L/day resulted in an accumulation of total VFA concentration up to 9.2 g/L as acetic acid (HAc) equivalent, causing rapid drops in pH and methane yield, and driving the digester to the verge of failure. Once intermittent (every 24 h) ORP-controlled micro-aeration (at ORP of -470 mV) was initiated, the total VFA concentration rapidly decreased to 3.0 g HAc/L and the methane yield improved, resulting in stable digester performance without the need for alkalinity supplementation or OLR reduction. By combining reactor performance results, mass balance analyses, microbial community characterization data, and a bioenergetic evaluation, this study suggested that rapid VFA conversion and CH4 production were carried out by facultative anaerobes and hydrogenotrophic methanogens under micro-aerobic conditions. This novel operating approach can be applied as an effective control strategy for high OLR AD processes especially in the event of VFA accumulation.

Biological strategies for enhanced hydrolysis of lignocellulosic biomass during anaerobic digestion: Current status and future perspectives.

Lignocellulosic biomass is the most abundant renewable bioresource on earth. In lignocellulosic biomass, the cellulose and hemicellulose are bound with lignin and other molecules to form a complex structure not easily accessible to microbial degradation. Anaerobic digestion (AD) of lignocellulosic biomass with a focus on improving hydrolysis, the rate limiting step in AD of lignocellulosic feedstocks, has received considerable attention. This review highlights challenges with AD of lignocellulosic biomass, factors contributing to its recalcitrance, and natural microbial ecosystems, such as the gastrointestinal tracts of herbivorous animals, capable of performing hydrolysis efficiently. Biological strategies that have been evaluated to enhance hydrolysis of lignocellulosic biomass include biological pretreatment, co-digestion, and inoculum selection. Strategies to further improve these approaches along with future research directions are outlined with a focus on linking studies of microbial communities involved in hydrolysis of lignocellulosics to process engineering.

Anaerobic biorefinery: Current status, challenges, and opportunities.

Anaerobic digestion (AD) has been in use for many decades. To date, it has been primarily aimed at treating organic wastes, mainly manures and wastewater sludge, and industrial wastewaters. However, with the current advancements, a more open mind is required to look beyond these somewhat restricted original applications of AD. Biorefineries are such concepts, where multiple products including chemicals, fuels, polymers etc. are produced from organic feedstocks. The anaerobic biorefinery concept is now gaining increased attention, utilizing AD as the final disposal step. This review aims at evaluating the potential significance of anaerobic biorefineries, including types of feedstocks, uses for the produced energy, as well as sustainable applications of the generated residual digestate. A comprehensive analysis of various types of anaerobic biorefineries has been developed, including both large-scale and household level applications. Finally, future directives are highlighted showing how anaerobic biorefinery concept could impact the bioeconomy in the near future.