Combining analytical approaches for better lignocellulosic biomass degradation: a way of improving fungal enzymatic cocktails?

PURPOSE: In this study, a combinatory approach was undertaken to assay the efficiency of fungal enzymatic cocktails from different fermentation conditions to degrade different lignocellulosic biomasses with the aim of finely characterizing fungal enzymatic cocktails. METHODS: Enzymatic assays (AZO and pNP-linked substrates and ABTS) were used to assess the composition of the fungal enzymatic cocktails for cellulase, xylanase and laccase activities. Comparisons were made with a new range of chromogenic substrates based on complex biomass (CBS substrates). The saccharification efficiency of the cocktails was evaluated as a quantification of the sugar monomers released from the different biomasses after incubation with the enzymatic cocktails. RESULTS: The results obtained showed striking differences between the AZO and pNP-linked substrates and the CBS substrates for the same enzymatic cocktails. On AZO and pNP-linked substrates, different hydrolysis profiles were observed between the different fungi species with Aspergillus oryzae being the most efficient. However, the results on CBS substrates were more contrasted depending on the biomass tested. Altogether, the results highlighted that assessing laccase activities and taking into account the complexity of the biomass to degrade were key in order to provide the best enzymatic cocktails. CONCLUSION: The complementary experiments performed in this study showed that different approaches needed to be taken in order to accurately assess the ability of an enzymatic cocktail to be efficient when it comes to lignocellulosic biomass degradation. The saccharification assay proved to be essential to validate the data obtained from both simple and complex substrates.

Optimized Bioproduction of Itaconic and Fumaric Acids Based on Solid-State Fermentation of Lignocellulosic Biomass.

The bioproduction of high-value chemicals such as itaconic and fumaric acids (IA and FA, respectively) from renewable resources via solid-state fermentation (SSF) represents an alternative to the current bioprocesses of submerged fermentation using refined sugars. Both acids are excellent platform chemicals with a wide range of applications in different market, such as plastics, coating, or cosmetics. The use of lignocellulosic biomass instead of food resources (starch or grains) in the frame of a sustainable development for IA and FA bioproduction is of prime importance. Filamentous fungi, especially belonging to the Aspergillus genus, have shown a great capacity to produce these organic dicarboxylic acids. This study attempts to develop and optimize the SSF conditions with lignocellulosic biomasses using A. terreus and A. oryzae to produce IA and FA. First, a kinetic study of SSF was performed with non-food resources (wheat bran and corn cobs) and a panel of pH and moisture conditions was studied during fermentation. Next, a new process using an enzymatic cocktail simultaneously with SSF was investigated in order to facilitate the use of the biomass as microbial substrate. Finally, a large-scale fermentation process was developed for SSF using corn cobs with A. oryzae; this specific condition showed the best yield in acid production. The yields achieved were 0.05 mg of IA and 0.16 mg of FA per gram of biomass after 48 h. These values currently represent the highest reported productions for SSF from raw lignocellulosic biomass.

Deciphering the role of Paenibacillus strain Q8 in the organic matter recycling in the acid mine drainage of Carnoulès.

BACKGROUND: The recycling of the organic matter is a crucial function in any environment, especially in oligotrophic environments such as Acid Mine Drainages (AMDs). Polymer-degrading bacteria might play an important role in such ecosystem, at least by releasing by-products useful for the rest of the community. In this study, physiological, molecular and biochemical experiments were performed to decipher the role of a Paenibacillus strain isolated from the sediment of Carnoulès AMD. RESULTS: Even though Paenibacillus sp. strain Q8 was isolated from an oligotrophic AMD showing an acidic pH, it developed under both acidic and alkaline conditions and showed a heterotrophic metabolism based on the utilization of a broad range of organic compounds. It resisted to numerous metallic stresses, particularly high arsenite (As(III)) concentrations (> 1,800 mg/L). Q8 was also able to efficiently degrade polymers such as cellulose, xylan and starch. Function-based screening of a Q8 DNA-library allowed the detection of 15 clones with starch-degrading activity and 3 clones with xylan-degrading activity. One clone positive for starch degradation carried a single gene encoding a "protein of unknown function". Amylolytic and xylanolytic activities were measured both in growing cells and with acellular extracts of Q8. The results showed the ability of Q8 to degrade both polymers under a broad pH range and high As(III) and As(V) concentrations. Activity measurements allowed to point out the constitutive expression of the amylase genes and the mainly inducible expression of the xylanase genes. PACE demonstrated the endo-acting activity of the amylases and the exo-acting activity of the xylanases. CONCLUSIONS: AMDs have been studied for years especially with regard to interactions between bacteria and the inorganic compartment hosting them. To date, no study reported the role of microorganisms in the recycling of the organic matter. The present work suggests that the strain Q8 might play an important role in the community by recycling the scarce organic matter (cellulose, hemicellulose, starch...), especially when the conditions change. Furthermore, function-based screening of a Q8 DNA library allowed to assign an amylolytic function to a gene previously unknown. AMDs could be considered as a reservoir of genes with potential biotechnological properties.

Evaluation of biological degradation of polyurethanes.

Polyurethanes (PU) are a family of versatile synthetic polymers intended for diverse applications. Biological degradation of PU is a blooming research domain as it contributes to the design of eco-friendly materials sensitive to biodegradation phenomena and the development of green recycling processes. In this field, an increasing number of studies deal with the discovery and characterization of enzymes and microorganisms able to degrade PU chains. The synthesis of short lifespan PU material sensitive to biological degradation is also of growing interest. Measurement of PU degradation can be performed by a wide range of analytical tools depending on the architecture of the materials and the biological entities. Recent developments of these analytical techniques allowed for a better understanding of the mechanisms involved in PU biodegradation. Here, we reviewed the evaluation of biological PU degradation, including the required analytics. Advantages, drawbacks, specific uses, and results of these analytics are largely discussed to provide a critical overview and support future studies.

Enzymatic recycling of thermoplastic polyurethanes: Synergistic effect of an esterase and an amidase and recovery of building blocks.

Biological recycling of polyurethanes (PU) is a huge challenge to take up in order to reduce a large part of the environmental pollution from these materials. However, enzymatic depolymerization of PU still needs to be improved to propose valuable and green solutions. The present study aims to identify efficient PU degrading enzymes among a collection of 50 hydrolases. Screenings based on model molecules were performed leading to the selection of an efficient amidase (E4143) able to hydrolyze the urethane bond of a low molar mass molecule and an esterase (E3576) able to hydrolyze a waterborne polyester polyurethane dispersion. Degradation activities of the amidase, the esterase and a mix of these enzymes were then evaluated on four thermoplastic polyurethanes (TPU) specifically designed for this assay. The highest degradation was obtained on a polycaprolactone polyol-based polyurethane with weight loss of 33% after 51 days measured for the esterase. Deep cracks on the polymer surface observed by scanning electron microscopy and the presence of oligomers on the remaining TPU detected by size exclusion chromatography evidenced the polymer degradation. Mixing both enzymes led to an increased amount of urethane bonds hydrolysis of the polymer. 6-hydroxycaproic acid and 4,4'-methylene dianiline were recovered after depolymerization as hydrolysis products. Such building blocks could get a second life with the synthesis of new macromolecular architectures.

Isolation and characterization of different promising fungi for biological waste management of polyurethanes.

As a highly resistant polymer family, polyurethanes (PU) are responsible for increasing environmental issues. Then, PU biodegradation is a challenging way to develop sustainable waste management processes based on biological recycling. Since the metabolic diversity of fungi is a major asset for polymer degradation, nearly thirty strains were isolated from sampling on six different PU wastes-containing environments. A screening of the fungi on four thermoplastic PU (TPU) with different macromolecular architectures led to the selection of three strains able to use two polyester PU as sole carbon source: Alternaria sp., Penicillium section Lanata-Divaricata and Aspergillus section flavi. Weight loss, FT-IR, Scanning Electron Microscopy and Size Exclusion Chromatography analyses revealed that these three fungi degrade slightly and similarly a fatty acid dimer-based TPU while variability of degradation was noticed on a polycaprolactone-based TPU. On this last TPU, robust analysis of the degraded polymers showed that the Penicillium strain was the best degrading microorganism. Membrane enzymes seemed to be involved in this degradation. It is the first time that a strain of Penicillium of the section Lanata-Divaricata displaying PU biodegradation ability is isolated. These newly discovered fungi are promising for the development of polyester PU waste management process.

Itaconic and Fumaric Acid Production from Biomass Hydrolysates by Aspergillus Strains.

Itaconic acid (IA) is a dicarboxylic acid included in the US Department of Energy's (DOE) 2004 list of the most promising chemical platforms derived from sugars. IA is produced industrially using liquid-state fermentation (LSF) by Aspergillus terreus with glucose as the carbon source. To utilize IA production in renewable resource-based biorefinery, the present study investigated the use of lignocellulosic biomass as a carbon source for LSF. We also investigated the production of fumaric acid (FA), which is also on the DOE's list. FA is a primary metabolite, whereas IA is a secondary metabolite and requires the enzyme cis-aconitate decarboxylase for its production. Two lignocellulosic biomasses (wheat bran and corn cobs) were tested for fungal fermentation. Liquid hydrolysates obtained after acid or enzymatic treatment were used in LSF. We show that each treatment resulted in different concentrations of sugars, metals, or inhibitors. Furthermore, different acid yields (IA and FA) were obtained depending on which of the four Aspergillus strains tested were employed. The maximum FA yield was obtained when A. terreus was used for LSF of corn cob hydrolysate (1.9% total glucose); whereas an IA yield of 0.14% was obtained by LSF of corn cob hydrolysates by A. oryzae.

Enzymatic cocktails produced by Fusarium graminearum under submerged fermentation using different lignocellulosic biomasses.

Fusarium graminearum was grown on four lignocellulosic substrates (corn cobs, wheat bran, hop cell walls, and birchwood) and glucose as the sole carbon source. Proteomic studies performed on the resulting enzymatic cocktails highlighted a great diversity in the number and type of proteins secreted. The cell wall-degrading enzymes (CWDE) proportion varied greatly from 20% to 69%. Only one of the 57 CWDEs detected in this study was common to the five proteomes. In contrast, 35 CWDEs were specific to one proteome only. The polysaccharide-degradation activities were different depending on the cocktail and the polysaccharide used. F. graminearum strongly modifies the enzymatic cocktail it secretes as a function of the biomass used for growth.