Lignocellulolytic Enzymes Research Articles

Biomass, lignocellulolytic enzyme production and lignocellulose degradation patterns by Auricularia auricula during solid state fermentation of corn stalk residues under different pretreatments

Current work proposes an innovative substitute cultivation strategy for Auricularia auricula that connects efficient lignocellulose biodegradation of corn stalk by pretreatment with mycelium growth of Auricularia auricula under solid state fermentation. The growth promotion effect of mycelium was as follows: alkali pretreatment > alkali combined with ozone pretreatment > ozone pretreatment > high-temperature water pretreatment (control). The highest cellulose degradation (35.38%) was observed in alkali pretreatment group (P < 0.05), while the maximum lignin loss of 28.10% was reached in the ozone pretreatment group. Correlation analysis showed that both cellulose and lignin could be used as carbon sources to promote mycelium growth, but the effect of cellulose was better. High activities of β-glucosidase (2.92 IU/g) and filter paper enzyme (2.92 IU/g) were observed in alkali pretreatment group on Day 6 and 8, respectively, which were significantly correlated with the change of cellulose degradation rate (P < 0.01).

Influence of carbon, nitrogen sources, inducers, and substrates on lignocellulolytic enzyme activities of Morchella spongiola

The influence of various carbon, nitrogen sources, inducers, and different agro-wastes on the growth and ligninolytic enzyme activities of Morchella spongiola (MR17) was studied. This fungus has grown well in mineral salts broth containing mannitol as the carbon and yeast extract as the nitrogen sources. In presence of inducers, the growth was inhibited compared to control. The carbon and nitrogen sources and the inducers affected the laccase activity of M. spongiola . Rhamnose and galactose supported the laccase activity more than other carbon sources tested. The manganese peroxidase (MnP) and versatile peroxidase (VP) activities were not detected in the medium amended with different carbon and nitrogen sources. The inducers, ABTS, and veratryl alcohol enhanced the laccase activity more than others. The MnP activity was maximum in wheat straw, followed by rice straw and phenol red. Maximum VP activity was observed in wheat straw, and rice straw, followed by veratryl alcohol. The protein content increased significantly in groundnuts shell and wheat grain substrates inoculated with M. spongiola . Wheat grains and wheat bran served as the most promising substrates with maximum lignocellulolytic activity. The laccase activity was higher in wheat grains and wheat bran, while VP and MnP activities were higher in rice straw and wheat straw. The cellulase activity was much less compared to other enzyme activities in all the substrates. Maximum lignin degradation was observed in wheat straw, followed by wheat bran. The cellulose degradation was significantly higher in groundnuts shell compared to other substrates. The present study results suggest that M. spongiola can utilize these substrates, induce enzyme activities, and have the potential for mushroom cultivation.

In Situ Corn Fiber Conversion for Ethanol Improvement by the Addition of a Novel Lignocellulolytic Enzyme Cocktail.

Corn mashes have high-viscosity and high-sugar characteristics, which hinders yeast-fermentation efficiency and the ethanol yield increase. The excessive viscosity of corn mash is caused by the unutilized cellulose in corn kernel fiber. A novel lignocellulolytic enzymes cocktail with strong substrate specificity was prepared for high-viscosity, high-sugar corn mash. The in situ conversion of corn mashes with novel lignocellulolytic enzymes at the optimum cellulase dosage of 50 FPU/L resulted in about 12% increased ethanol concentration compared with the reference mash at different batch-fermentation scales. Adding the lignocellulolytic enzymes caused the greatest decrease in viscosity of corn mash and residual sugars by 40.9% and 56.3%, respectively. Simultaneously, the application of lignocellulolytic enzymes increased the value of the dried distiller’s grain with solubles (DDGS) by increasing the protein content by 5.51%. The in situ conversion of cellulose can decrease the fermentation broth viscosity and improve the rheological property, thereby improving the ethanol yield. With the same amount of material, the application of the novel enzymes cocktail can enhance the ethanol yield by more than 12%. A quarter of the ethanol yield increase was due to the further hydrolysis of starch, while three quarters to cellulose. Thus, this technology will increase the net revenue of bioethanol industrialization.

GlSwi6 Positively Regulates Cellulase and Xylanase Activities through Intracellular Ca2+ Signaling in Ganoderma lucidum.

Ganoderma lucidum is a white-rot fungus that produces a range of lignocellulolytic enzymes to decompose lignin and cellulose. The mitogen-activated protein kinase (MAPK) pathway has been implicated in xylanases and cellulases production. As the downstream transcription factor of Slt2-MAPK, the function of Swi6 in G. lucidum has not been fully studied. In this study, the transcription factor GlSwi6 in G. lucidum was characterized and shown to significantly positively regulate cellulases and xylanases production. Knockdown of the GlSwi6 gene decreased the activities of cellulases and xylanases by approximately 31%~38% and 54%~60% compared with those of the wild-type (WT) strain, respectively. Besides, GlSwi6 can be alternatively spliced into two isoforms, GlSwi6A and GlSwi6B, and overexpression of GlSwi6B increased the activities of cellulase and xylanase by approximately 50% and 60%, respectively. Further study indicates that the existence of GlSwi6B significantly increased the concentration of cytosolic Ca2+. Our study indicated that GlSwi6 promotes the activities of cellulase and xylanase by regulating the Ca2+ signaling. These results connected the GlSwi6 and Ca2+ signaling in the regulation of cellulose degradation, and provide an insight for further improvement of cellulase or xylanase activities in G. lucidum as well as other fungi.

Characterization of lignocellulolytic fungi isolated from cocoa rhizosphere under different cropping patterns

Cocoa plantations may provide a considerable amount of fungal biodiversity with high potential as soil organic matter biodecomposer agents. Therefore, the isolation and characterization of potential fungi is the first step to obtain the wide variety of lignocellulolytic enzymes. This study aimed at identifying potential lignocellulolytic fungi isolated from different type cocoa cropping pattern. The lignocellulose-degrading enzymes was determined by growing the isolated fungi on Guaiacol containing PDA media, and CMC media with additional of Red Congo solution. Their potential index (IP) was scored by measuring the diameter of the clear zone around the fungal colonies. Our identifications suggest the presence of both ligninolytic, and cellulolytic enzymes through a reddish, and clear zones produced around the colonies. Additionally, we identified two types of potential fungi with higher potential index both ligninolytic and cellulolytic, including strain fungal CSF#5 and CSF#15 which belongs to the group of fungi Ascomycetes.

Lignocellulolytic enzyme potential of dark septate endophyte (dse) from Pinus merkusii roots in Dramaga Bogor Indonesia

The ability of Dark Septate Endophyte (DSE) derived from the roots of P. merkusii in decomposing organic matter is not much published. This study aims to determine the activity of lignolytic and cellulolytic enzymes, as well as the value of the cellulolytic index of Apls, isolates from P. merkusii roots taken from IPB Dramaga Bogor. Enzyme activity was tested by measuring the clear zone formed, while the cellulolytic index value was obtained by measuring the ratio of the clear zone formed around the colony. Data were analyzed descriptively. The results identified 10 isolates, 8 Apls isolates had potential enzyme activity that able to form clear zone, 5 isolates had lignolytic enzyme activity (Apls 1.4.1, Apls 2.3.1.1, Apls 3.4.2, Apls 2.1.1 and Apls 3.4 1.1) and 1 isolate had cellulolytic enzyme activity (Apls 3.1.3), and 2 isolates had lignocellulolytic enzyme activity (Apls 1.5.3 and Apls 3.4.3b), with the highest cellulolytic index value in Apls 1.5.3 isolates of 0,87 mm. 2 isolates had not lignocellulolytic enzymes activity (Apls 3.4.3 and Apls 3.1.4). The DSE roots of P. merkusii from IPB Dramaga Bogor had the potential for lignocellulolytic enzymes of 20 per cent of the total DSE found.

Effect of barley straw and Egyptian clover hay on the rumen fermentation and structure and fibrolytic activities of rumen bacteria in dromedary camel.

Background and Aim:Understanding the regulations of rumen microbiota and their fibrolytic capabilities under different forages are essential to improve rumen fermentation and animal feed efficiency. This study aimed to evaluate the changes in the rumen fermentation and the structure and fibrolytic activities of rumen bacteria in camels fed barley straw and Egyptian clover hay.Materials and Methods:Three fistulated camels were fed a diet containing barley straw for 30 days; then transitioned to a diet containing Egyptian clover hay for 30 days. In addition, bacterial media enriched with xylan and different cellulose sources, namely, filter paper, wheat straw, and alfalfa hay, were used to evaluate the ability of camel rumen bacteria to produce xylanase and cellulase enzymes.Results:The camel group fed Egyptian clover hay showed higher crude protein intake, rumen ammonia, total volatile fatty acids, and acetic acid. Moreover, the camel group fed barley straw showed higher neutral detergent fiber intake, rumen pH, and propionic and butyric acids. Principal component analysis showed that bacterial communities were separated based on the forage type. Forage type affected the composition of rumen bacteria and most of the bacterial community was assigned to phylum Bacteroidetes and Firmicutes. Egyptian clover hay diet increased the proportions of genus Prevotella and Ruminococcus; while fed barley straw diet increased the Butyrivibrio, RC9_gut_group, and Fibrobacteres. The bacterial culture of the Egyptian clover hay fed group produced the greatest xylanase and the bacterial culture of the barley straw fed group produced the maximum cellulase.Conclusion:Egyptian clover hay is recommended to feed camels in intensive production. Moreover, the bacterial community in the camel rumen is a promising source of lignocellulolytic enzymes.

Enhancement of mycelial biomass and lignocellulolytic enzymes of milky mushroom Calocybe indica through supplementation with organic amendments

Milky mushroom (Calocybe indica) also known as tropical mushroom or summer mushroom has significant attributes of milky white colour with high nutritive value and long shelf life. The yield performance of milky mushrooms again depends on the secretion of lignocellulolytic enzymes that further influence the colonization of mycelium on the substrates. In the present study, organic supplements viz. cakes of cotton, groundnut, sesamum, coconut cake, neem cake, powdered moth bean, red gram, horse gram, green gram, soya bean, lab-lab, maize, rice bran and wheat bran amended at 1% and 5% concentration induced profuse mycelial growth and enhanced ligninolytic enzyme secretion by Calocybe indica under in vitro conditions. The results revealed that wheat bran and red gram supplements at 5 % concentration in PDA medium were found to influence the mycelial growth on the 7th day (68.67 mm and 62.33 mm respectively). Similarly, both wheat bran and red gram supplements at 5 % in liquid medium supported increase in the biomass production of C. indica on the 15th day (1.83 and 1.40 g) followed by horse gram. The influence of organic supplements on the induction of lignocellulolytic activity showed that the maximum cellulase and xylanase activity (1.7 U/min/g and 0.835 U/min/g respectively) and laccase activity (1.6 U/min/g) were noticed in the substrate supplemented with wheat bran followed by horse gram (1.3 U/min/g) at 5% concentration under solid-state fermentation. However, the lignin peroxidase activity was maximum (0.59 U/min/g) in combination with wheat bran (2.5%) and red gram (2.5%) and manganese peroxidase activity was maximum in wheat bran supplement (0.3 U/min/g) at 5% concentration. It is inferred that amending organic supplements viz. wheat bran, horse gram and red gram to substrates enhance the mycelial growth, biomass production and lignocellulolytic enzyme secretion of C. indica and offers scope for yield improvement.

Thermostable Cellulases / Xylanases From Thermophilic and Hyperthermophilic Microorganisms: Current Perspective.

The bioconversion of lignocellulose into monosaccharides is critical for ensuring the continual manufacturing of biofuels and value-added bioproducts. Enzymatic degradation, which has a high yield, low energy consumption, and enhanced selectivity, could be the most efficient and environmentally friendly technique for converting complex lignocellulose polymers to fermentable monosaccharides, and it is expected to make cellulases and xylanases the most demanded industrial enzymes. The widespread nature of thermophilic microorganisms allows them to proliferate on a variety of substrates and release substantial quantities of cellulases and xylanases, which makes them a great source of thermostable enzymes. The most significant breakthrough of lignocellulolytic enzymes lies in lignocellulose-deconstruction by enzymatic depolymerization of holocellulose into simple monosaccharides. However, commercially valuable thermostable cellulases and xylanases are challenging to produce in high enough quantities. Thus, the present review aims at giving an overview of the most recent thermostable cellulases and xylanases isolated from thermophilic and hyperthermophilic microbes. The emphasis is on recent advancements in manufacturing these enzymes in other mesophilic host and enhancement of catalytic activity as well as thermostability of thermophilic cellulases and xylanases, using genetic engineering as a promising and efficient technology for its economic production. Additionally, the biotechnological applications of thermostable cellulases and xylanases of thermophiles were also discussed.

EFFECT OF SOME AGRO-INDUSTRIAL RESIDUES ON MYCELIAL GROWTH AND PRODUCTION OF LIGNOCELLULOLYTIC ENZYMES BY A FUNGUS NATIVE TO ALGERIAN FOREST

Valorization of agricultural and agro-food by-products by fermentation constitutes a very interesting biotechnological approach for the production of lignocellulolytic enzymes. This work was carried out to reveal the effect of some lignocellulosic materials on the mycelial growth and lignocellulolytic enzymes production by Bjerkandera adusta BRFM 1916. The strain showed ABTS- and guaiacol-oxidation activities. The optimal temperature for mycelial growth was 28 °C. The maximum growth rate of this fungus was achieved on wheat bran (2.08 ± 0.05 cm day-1), followed by barley bran, with a significant reduction of 6.73%. Several agricultural lignocellulosic residues were used as substrates for enzymes production. All the data indicated differential utilization of the various materials by the fungus. The selected fungus produced good CMCase (690 ± 0.066 UL-1) and β-Glu (253 UL-1) activities on wheat bran and orange peels, respectively. A high level of MnP activity (449.21 ± 3.44 UL-1) was obtained on wheat bran.

Characterization and modulation of endoplasmic reticulum stress response target genes in Kluyveromyces marxianus to improve secretory expressions of heterologous proteins

BackgroundKluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. Expressions of heterologous proteins usually lead to the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER) and then cause ER stress. To cope with this problem, a group of ER stress response target genes (ESRTs) are induced, mainly through a signaling network called unfolded protein response (UPR). Characterization and modulation of ESRTs direct the optimization of heterologous expressions. However, ESRTs in K. marxianus have not been identified so far.ResultsIn this study, we characterized the ER stress response in K. marxianus for the first time, by using two ER stress-inducing reagents, dithiothreitol (DTT) and tunicamycin (TM). Results showed that the Kar2–Ire1–Hac1 pathway of UPR is well conserved in K. marxianus. About 15% and 6% of genes were upregulated during treatment of DTT and TM, respectively. A total of 115 upregulated genes were characterized as ESRTs, among which 97 genes were identified as UPR target genes and 37 UPR target genes contained UPR elements in their promoters. Genes related to carbohydrate metabolic process and actin filament organization were identified as new types of UPR target genes. A total of 102 ESRTs were overexpressed separately in plasmids and their effects on productions of two different lignocellulolytic enzymes were systematically evaluated. Overexpressing genes involved in carbohydrate metabolism, including PDC1, PGK and VID28, overexpressing a chaperone gene CAJ1 or overexpressing a reductase gene MET13 substantially improved secretion expressions of heterologous proteins. Meanwhile, overexpressing a novel gene, KLMA_50479 (named ESR1), as well as overexpressing genes involved in ER-associated protein degradation (ERAD), including HRD3, USA1 andYET3, reduced the secretory expressions. ESR1 and the aforementioned ERAD genes were deleted from the genome. Resultant mutants, except the yet3Δ mutant, substantially improved secretions of three different heterologous proteins. During the fed-batch fermentation, extracellular activities of an endoxylanase and a glucanase in hrd3Δ cells improved by 43% and 28%, respectively, compared to those in wild-type cells.ConclusionsOur results unveil the transcriptional scope of the ER stress response in K. marxianus and suggest efficient ways to improve productions of heterologous proteins by manipulating expressions of ESRTs.

Overview of Lignocellulolytic Enzyme Systems with Special Reference to Valorization of Lignocellulosic Biomass.

Lignocellulosic biomass, one of the most valuable natural resources, is abundantly present on earth. Being a renewable feedstock, it harbors a great potential to be exploited as a raw material, to produce various value-added products. Lignocellulolytic microorganisms hold a unique position regarding the valorization of lignocellulosic biomass as they contain efficient enzyme systems capable of degrading this biomass. The ubiquitous nature of these microorganisms and their survival under extreme conditions have enabled their use as an effective producer of lignocellulolytic enzymes with improved biochemical features crucial to industrial bioconversion processes. These enzymes can prove to be an exquisite tool when it comes to the eco-friendly manufacturing of value-added products using waste material. This review focuses on highlighting the significance of lignocellulosic biomass, microbial sources of lignocellulolytic enzymes and their use in the formation of useful products.

Sustainable Production of Lignocellulolytic Enzymes in Solid-State Fermentation of Agro-Industrial Waste: Application in Pumpkin (Cucurbita maxima) Juice Clarification

Valorization of agro-industrial waste through greener and biotechnological processes are promising approaches to minimize the generation of agro-industrial waste. Therefore, the study aimed to produce lignocellulolytic enzymes from agro-industrial waste under solid-state fermentation (SSF) conditions and study their application in the clarification of pumpkin juice. The SSF was performed with three different combinations of wheat bran + rice bran (WBRB), wheat bran + wheat straw (WBWS), and rice bran + wheat straw (RBWS) as dry solid substrates (1:1) using Fusarium oxysporum (MTCC 7229). The protein, carboxymethyl cellulase (CMCase), and xylanase contents ranged from 0.98–3.90 mg/g, 5.89–6.84 U/g substrate, and 10.08–13.77 U/g substrate, respectively in different agro-industrial waste as substrates (WBRB, WBWS, RBWS, and control). The increase in enzyme concentration (0.50–2.40%) added to pumpkin juice exhibited an increased juice yield (16.30–55.60%), reduced browning index (1.03–0.70), and an increase in clarity (5.31–13.77 %T), which was further confirmed by a total variance of 84.83% by principal component analysis. Thus, the low-cost lignocellulolytic enzymes can be produced from agro-industrial waste that might have applications in food and beverage industries. Hence, this approach could be used as a long-term sustainable and circular source to valorize agro-industrial waste towards the greener future and the preservation of ecosystems.

Towards the Discovery of Active Lignocellulolytic Enzyme Producers: A Screening Study of Xylotrophic Macrofungi from the Central Russian Upland

Towards the Discovery of Active Lignocellulolytic Enzyme Producers: A Screening Study of Xylotrophic Macrofungi from the Central Russian Upland

Comprehensive strategies of Lignocellulolytic enzyme production from microbes and their applications in various commercial-scale faculties

Activities of anthropological organisms lead to the production of massive lignocellulosic waste every year and these lignocellulolytic enzymes plays crucial role in developing eco-friendly, sustainable and economical methods for decomposing and pre-treating the biomass to produce biofuels, organic acids, feeds and enzymes. Lignocellulolytic enzymes sustainably hydrolyse the biomass and can be utilized in wide range of applications such as personal care, pharmaceutical, biofuel release, sewage treatment, food and beverage industries. Every year a significant ton of biomass waste is released and insight on these crucial enzymes could establish in all the industries. However, due to the increased demand for compost materials, biomass degradation has resulted in composting processes. Several methods for improving compost amount and quality have been explored, including increasing decomposer inoculums, stimulating microbial activity, and establishing a decomposable environment. All of these prerequisites are met by biotechnological applications. Biotechnological procedures are used to improve the activity of enzymes on biomass. It leads to an adequate supply of compost and base materials for enterprises. In terms of effectiveness and stability during the breakdown process, lignocellulolytic enzymes derived from genetically modified species outperformed naturally derived lignocellulolytic enzymes. It has the potential to increase the quality and output of by-products. This review discussed the development of lignocellulolytic enzyme families and their widespread applications in a variety of industries such as olive oil extraction, carotenoid extraction, waste management, pollution control, second-generation bio-ethanol production, textile and dyeing, pharmaceuticals, pulp and paper, animal feed, food processing industries, detergent, and agricultural industries.

Taxonomic structure and function of the corn stover degradative microbial consortium GF‐20 following growth on different sources of nitrogen

AbstractMicrobial consortia have substantial promise to degrade returned straw biomass and implement conversion strategies for soil nutrients to improve the quality of cultivated land. However, the composition of microbial consortia under different nitrogen conditions, the interaction between members and the functions during the process of decomposition of lignocellulose remain poorly understood. This study comprehensively examines the functional potential and structural diversity of the GF‐20 microbial consortium that degrades lignocellulose and soil used for inoculation and will explore the associated metabolic pathway networks in Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) using metagenomic analyses. Analyses of the metagenomic taxonomic affiliation data showed that the soil used for inoculation primarily contained members from the phyla Proteobacteria, Bacteroidetes, Acidobacteria, Chloroflexi and Acidobacteria, as well as Proteobacteria and Bacteroidetes in microbial consortia (N1 and N6). However, N6 is likely to be able to reduce the diversity of microbial species and change the community structure in a more concentrated comparison with N1. Notably, the key microbes were determined to be Cellvibrio, Brevundimonas, Flavobacterium and Achromobacter, whose analogous physiological functions were critical for the degradation of lignocellulosic substrates. The COG annotation indicated that the microbial metabolic cluster was the predominant category in all the samples, while the metabolic genes in N6 were relatively less abundant compared with those in N1. The KEGG annotation demonstrated that the inoculation soil (Q) has more metabolic functions and pathways than those of the microbial consortia, and the number of orthologies, modules, pathways and enzymes in N6 was observably lower than that in N1. A more detailed analysis showed that both of the consortia had functional profiles that were highly similar. These functional profiles clearly differed compared with those in the Q community. The relative abundance (RA) of ABC transporters in the Q was higher than that in the microbial consortia and higher in N1 than in N6. However, the two‐component system of N1 and N6 was higher than that of Q because of their relation to the microbial degradation of lignocellulosic materials. Most of the abundances of lignocellulolytic enzymes in the Q were lower than those in the microbial consortia (N1 and N6), and the RAs of some of these enzymes (e.g., β‐glucosidase, xylan 1,4‐β‐xylosidase, arabinoxylan arabinofuranohydrolase, β‐galactosidase, feruloyl esterase, mannan endo‐1,4‐β‐mannosidase, alpha‐glucuronidase and arabinogalactan endo‐1,4‐β‐galactosidase) in N6 were significantly higher than those of N1.The taxonomic structure and function of the Q and that of the consortia differed significantly, and the use of urea resulted in a decrease in the taxonomic species of microorganisms. The changes in functional diversity were accompanied with variation in the microbial composition, many of which were related to the microbial degradation of lignocellulolytic materials. Most of the degradative lignocellulolytic function in Q was lower than those of the microbial consortia N1 and N6, which aid in the degradation of straw.

Bioprospecting of microorganisms with lignocellulolytic enzyme activity for leaf litter degradation

This study aims to isolate cellulose and lignin degrading microorganisms which are capable of degrading lignocellulosic biomass in an ecofriendly nature. A total of 160 cultures were isolated and screened for their cellulolytic, ligninolytic activity. Those cultures were forwarded for lignocellulolytic enzyme assays. Among the bacterial isolate, TER3 showed the maximum number of enzyme activity in cellulase, PPO and laccase (6.024 U/ml, 0.963 U/ml and 0.215 U/ml respectively). E-PW1 isolates were observed, to the production of PPO (0.237 U/ml), MnP (0.007 U/ml) and LiP (0.242 U/ml). E-MUS1 isolate also showed good activity in cellulase (2.875 U/ml), LiP (0.240 U/ml) production. In the case of fungal isolates TER1 exhibited the highest activity in PPO (2.468 U/ml), laccase (0.053 U/ml) and LiP (0.244 U/ml) production, COM3 isolate recorded activity of 0.490 U/ml in PPO and 0.240 U/ml in LiP followed by E-PST3 (0.013U/ml in laccase, 0.203U/ml in LiP). In Actinobacterial culture CP4 showed higher activity in cellulase production (5.140 U/ml), laccase (0.124 U/ml) production, E-PST4 showed 0.008 U/ml in laccase, 0.149U/ml in LiP production and COM2 in cellulase (2.66U/ml). Selection of cultures were undertaken based on the number of enzymes produced. The selected 9 cultures were considered as the best lignocellulose degrading microorganism that could produce all the desired enzymes for effective degradation of lignocellulosic biomass.

Characterisation of the enzyme transport path between shipworms and their bacterial symbionts

BackgroundShipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal’s gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood.ResultsThrough a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system.ConclusionBased on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm’s mouth and digestive tract, where they aid in wood digestion.

Responses of Soil Microbial Population and Lignocellulolytic Enzyme Activities to Palm Kernel Shell Biochar Amendment

Responses of Soil Microbial Population and Lignocellulolytic Enzyme Activities to Palm Kernel Shell Biochar Amendment

Screening of Lignocellulolytic Enzyme Activities in Fungal Species and Sequential Solid-State and Submerged Cultivation for the Production of Enzyme Cocktails.

Various fungal species can degrade lignocellulolytic materials with their enzyme cocktails composed of cellulolytic and lignolytic enzymes. In this work, seven fungal species (Mucor indicus DSM 2185, Paecilomyces variotii CBS 372.70, Myceliophthora thermophila CBS 663.74, Thielavia terrestris CBS 456.75, Botryosphaeria dothidea JCM 2738, Fusarium oxysporum f.sp. langenariae JCM 9293, and Fusarium verticillioides JCM 23107) and four nutrient media were used in the screening for effective lignocellulose degrading enzymes. From the seven tested fungi, F. oxysporum and F. verticilliodes, along with nutrient medium 4, were selected as the best medium and producers of lignocellulolytic enzymes based on the determined xylanase (>4 U mg−1) and glucanase activity (≈2 U mg−1). Nutrient medium 4 supplemented with pretreated corn cobs was used in the production of lignocellulolytic enzymes by sequential solid-state and submerged cultivation of F. oxysporum, F. verticilliodes, and a mixed culture of both strains. F. oxysporum showed 6 times higher exoglucanase activity (3.33 U mg−1) after 5 days of cultivation in comparison with F. verticillioides (0.55 U mg−1). F. oxysporum also showed 2 times more endoglucanase activity (0.33 U mg−1). The mixed culture cultivation showed similar endo- and exoglucanase activities compared to F. oxysporum (0.35 U mg−1; 7.84 U mg−1). Maximum xylanase activity was achieved after 7 days of cultivation of F. verticilliodes (≈16 U mg−1), while F. oxysporum showed maximum activity after 9 days that was around 2 times lower compared to that of F. verticilliodes. The mixed culture achieved maximum xylanase activity after only 4 days, but the specific activity was similar to activities observed for F. oxysporum. It can be concluded that both fungal strains can be used as producers of enzyme cocktails for the degradation of lignocellulose containing raw materials, and that corn cobs can be used as an inducer for enzyme production.