Physiology and enzymology of lignocellulose digestion in the shipworm Lyrodus pedicellatus

Shipworms are marine bivalve molluscs, known for their wood boring abilities. They use modified shells to bore into and grind wood, which is then digested. The shipworm’s ability to feed on lignocellulose is dependent on the presence of endosymbiotic bacteria that live in the animal’s gills inside specialized eukaryotic cells called bacteriocytes. These bacteria provide the animal with hydrolytic enzymes for wood digestion, which are translocated from the gills to the caecum, the main site of wood digestion. Unlike other lignocellulose degrading organisms, which harbour symbiotic microbes in their digestive tract, the shipworm caecum hosts only few bacteria but contains a large amount of carbohydrate active enzymes (CAZymes) of both endogenous and bacterial origin. This study investigates the anatomical, physiological and molecular basis of wood digestion in the shipworm Lyrodus pedicellatus. A combination of meta-transcriptomics, meta-proteomics and microscopic studies of the shipworm digestive organs were used, coupled with recombinant production and characterisation of some of the most expressed lignocellulolytic enzymes. This multidisciplinary analysis revealed how two structures, the food groove (a mucus stream utilised by filter feeding molluscs to transport food particles from the gills to the digestive system) and the crystalline style (a rotating structure hosted in the stomach involved in extra-cellular digestion) have been co-opted in shipworms to translocate bacteria and their enzymes from the gills to the caecum, to facilitate wood digestion. The transcriptomic and proteomic results indicate that bacterial lignocellulolytic enzymes are expressed in the gills, while the endogenous enzymes are mainly produced by the digestive glands, with complementary CAZy classes being expressed by the bacteria and the shipworms, indicating a subdivision of roles. Five bacterial CAZymes were recombinantly expressed and characterised, showing activity on cellulose, galacto-glucomannan and xylan. This study provides new insights into the mechanisms of wood digestion in shipworms, which may have biotechnological relevance

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

BACKGROUND: Shipworms 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. RESULTS: Through 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. CONCLUSION: Based 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

Uncovering the molecular mechanisms of lignocellulose digestion in shipworms

Abstract Lignocellulose forms the structural framework of woody plant biomass and represents the most abundant carbon source in the biosphere. Turnover of woody biomass is a critical component of the global carbon cycle, and the enzymes involved are of increasing industrial importance as industry moves away from fossil fuels to renewable carbon resources. Shipworms are marine bivalve molluscs that digest wood and play a key role in global carbon cycling by processing plant biomass in the oceans. Previous studies suggest that wood digestion in shipworms is dominated by enzymes produced by endosymbiotic bacteria found in the animal’s gills, while little is known about the identity and function of endogenous enzymes produced by shipworms. Using a combination of meta-transcriptomic, proteomic, imaging and biochemical analyses, we reveal a complex digestive system dominated by uncharacterized enzymes that are secreted by a specialized digestive gland and that accumulate in the cecum, where wood digestion occurs. Using a combination of transcriptomics, proteomics, and microscopy, we show that the digestive proteome of the shipworm Lyrodus pedicellatus is mostly composed of enzymes produced by the animal itself, with a small but significant contribution from symbiotic bacteria. The digestive proteome is dominated by a novel 300 kDa multi-domain glycoside hydrolase that functions in the hydrolysis of β-1,4-glucans, the most abundant polymers in wood. These studies allow an unprecedented level of insight into an unusual and ecologically important process for wood recycling in the marine environment, and open up new biotechnological opportunities in the mobilization of sugars from lignocellulosic biomass

An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion.

Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia's LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases

Hemocyanin facilitates lignocellulose digestion by wood-boring marine crustaceans

Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for innovations in lignocellulose biorefining

Recovery of bio-based products from PHA-rich biomass obtained from biowaste: A review

PHAs produced from biowaste are an ecological alternative to petroleum-based plastics. Despite their advantageous characteristics, their commercialisation is limited because the extraction process is still inefficient. This review describes approaches applied to PHA-rich biomass to recover bio-based products beyond their valorisation as bioplastics. The whole PHA-containing microbial cell can be used for animal feed, since PHAs can boost the immune system of the fed animals, and direct extrusion of PHA-rich biomass allows the production of biocomposites. After PHAs extraction, by-products can be recovered to make high-value products such as animal feed, biostimulants, protein hydrolysates, flocculants, adhesives and pharmaceutical. PHAs-rich microbes can also be fed to animals able to excrete PHAs in their faecal pellets, from which they can be easily retrieved. Such approaches have the potential to favour the transition to bioplastics obtained from biowaste streams, thus reducing the environmental impact of plastic production

Biological Conversion of Agricultural Wastes Into Microbial Proteins for Aquaculture Feed

The growing world population will comprise 9 billion people by 2050 and protein consumption will be 50% higher in 2030 compared to 2000. To prepare for this increase, new sources of protein must be sought to replace the non-environmentally sustainable animal and plant proteins that are currently produced. Insects or microbial proteins can indeed be an economic and nutritious alternative not only for animal feed, but also as supplements for human consumption. Our study aimed at assessing the production of microbial proteins destined to aquaculture feed, using as starting material agricultural and animal wastes, in order to tackle at the same time the problem of waste disposal. A bioreactor was feed with the fluid obtained from the acidogenic fermentation of zootechnical and agricultural waste, containing a mixture of VFAs of which acetic acid was the most abundant (43.8 %). The highest productivity (1.6-2.0 gMLVSS/L per day) was obtained with an HRT of 2 days and an ORL of 22 gCOD/L per day. The produced bacterial biomass was analyzed to determine its suitability as fish feed, showing a crude protein content of 73 % over the MLVSS, of which glutamic and aspartic acid were the most abundant. These amounts are higher than what is found in most commercial fish feeds, suggesting that the bacterial biomass has potential to be used as aquaculture feed. PHAs accumulation tests on part of the biomass recovered from the bioreactor showed concentration of PHAs up to 62.2 % over cell dry weight, a positive result since PHAs have the potential to enhance fish growth

Upcycling of PHA-producing bacteria for biostimulants production and polyhydroxyalkanoates recovery

The use of petroleum-based plastic has led to its accumulation in the environment, with negative impacts on the eco- system and the biota. Polyhydroxyalkanoates (PHAs), biobased and biodegradable plastics produced by microbes, have many commercial applications, however their high production cost means they cannot yet compete with tradi- tional plastics. At the same time, the problem of the growing human population implies that improved crop production is needed to avoid malnutrition. Biostimulants enhance plant growth and have the potential to improve agricultural yields; they can be obtained from biological feedstock, including microbes. Therefore, there is scope for coupling the production of PHAs with that of biostimulants, making the process more cost-efficient and minimizing by- products generation. In this work, low-value agro-zoological residues were processed to obtain PHA-storing bacteria via acidogenic fermentation; PHAs destined for the bioplastic market were extracted, and the protein-rich by- products were turned into protein hydrolysates using different treatment methods, assessing their biostimulant effects in growth trials with tomato and cucumber plants. The results indicate that the best hydrolysis treatment, realizing the highest amount of organic nitrogen (6.8 gN-org/L) while achieving the best PHA recovery (63.2 % gPHA/gTS), is ob- tained with strong acids. All the protein hydrolysates were effective in improving either roots or leaf development, with various results, depending on the species and the growth method. The acid hydrolysate was the most effective treatment to enhance the development of shoots (21 % increase compared to the control) and roots (16 % increase for the dry weight and 17 % for main root length) of hydroponically-grown cucumber plants, while pot-grown toma- toes, biostimulated via foliar spray, developed bigger shoots (up to 41 %) with the hydrolysate obtained from the al- kaline treatment. These preliminary results indicate that simultaneous production of PHAs and biostimulants is feasible, and that commercialization could be achievable given the expected reduction in production costs

Biological conversion of agricultural residues into microbial proteins for aquaculture using PHA-producing mixed microbial cultures

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Production and characterization of PHAs by pure culture using protein hydrolysates as sole carbon source

Protein hydrolysates obtained from discarded biomass can be further upgraded into high market value products, in the optic of a circular bioeconomy. In this work, residues from the cultivation of alfalfa, soybean and rice, and bovine wet blue shavings were fermented with mixed microbial cultures obtaining high concentrations of volatile fatty acid (up to 50 times compared to the original hydrolysate), mainly butyric and acetic acid. This rich medium was used for growing the bacterium Thauera sp., a known producer of polyhydroxyalkanoates (PHAs), biodegradable polymers with potential to replace petrol-based plastics. The overall process resulted in the production of 1.4 gPHAs/L, with a conversion rate of 32% for the alfalfa hydrolysates when considering the COD given by the initial VFAs. The obtained biopolymer was poly(3-hydroxybutyrate-co-3-hydroxyvalerate), as confirmed by the presence of characteristic peaks and by the melting temperatures and thermo-oxidative degradation in the expected range; the polymer has a high degree of purity, being without inorganic residues. This work showed the feasibility of a process aimed at the valorisation of protein hydrolysates into high-market value products such as bioplastics