Complex Sugars Research Articles

Bacterial Exopolysaccharides: From Production to Functional Features

Exopolysaccharides, known as bacterial EPS, are complex sugar polymers that bacteria secrete into their environment. EPSs play a crucial role in bacterial survival and proliferation by protecting cells from environmental threats. They also contribute to proper adhesion to numerous surfaces through biofilm production. Bacterial EPS display a wide range of biological activities conferred by their outstanding physical and chemical properties, which make them great candidates for medical and industrial applications. The main biological activities recorded up-to-day include antioxidant, anticancer, antiviral, anti-inflammatory, anticancer, antibacterial, immune-modulatory and chelating properties. They also can be used as thickening agents in the industry or as additives to improve soil quality in agriculture. The current review offers a thorough overview of bacterial EPS, their biosynthesis processes and regulation, and biotechnological strategies to increase their production. The review also unravels the main extraction, purification, and identification techniques and highlights the key functional features of these complex molecules.

Computational Study on the Water Corrosion Process at Schreibersite (Fe2NiP) Surfaces: from Phosphide to Phosphates.

Phosphorus (P) is a fundamental element for whatever form of life, in the same way as the other biogenic macroelements (SONCH). The prebiotic origin of P is still a matter of debate, as the phosphates present on earth are trapped in almost insoluble solid matrixes (apatites) and, therefore, hardly available for inclusion in living systems in the prebiotic era. The most accepted theories regard a possible exogenous origin during the Archean Era, through the meteoritic bombardment, when tons of reactive P in the form of phosphide ((Fe,Ni)3P, schreibersite mineral) reached the primordial earth, reacting with water and providing oxygenated phosphorus compounds (including phosphates). In the last 20 years, laboratory experiments demonstrated that the corrosion process of schreibersite by water indeed leads to reactive phosphates that, in turn, react with other biological building blocks (nucleosides and simple sugars) to form more complex molecules (nucleotides and complex sugars). In the present paper, we study the water corrosion of different crystalline surfaces of schreibersite by means of periodic DFT (density functional theory) simulations. Our results show that water adsorbs molecularly on the most stable (110) surface but dissociates on the less stable (001) one, giving rise to further reactivity. Indeed, subsequent water adsorptions, up to the water monolayer coverage, show that, on the (001) surface, iron and nickel atoms are the first species undergoing the corrosion process and, in a second stage, the phosphorus atoms also get involved. When adsorbing up to three and four water molecules per unit cell, the most stable structures found are the phosphite and phosphate forms of phosphorus, respectively. Simulation of the vibrational spectra of the considered reaction products revealed that the experimental band at 2423 cm-1 attributed to the P-H stretching frequency is indeed predicted for a phosphite moiety attached to the schreibersite (001) surface upon chemisorption of up to three water molecules.

Martini-3 Coarse-Grained Models for the Bacterial Lipopolysaccharide Outer Membrane of Escherichia coli.

The outer lipopolysaccharide (LPS) membrane of Gram-negative bacteria forms the main barrier for transport of antimicrobial molecules into the bacterial cell. In this study we develop coarse-grained models for the outer membrane of Escherichia coli in the Martini-3 framework. The coarse-grained model force field was parametrized and validated using all-atom simulations of symmetric membranes of lipid A and rough LPS as well as a complete asymmetric membrane of LPS with the O-antigen. The bonded parameters were obtained using an iterative refinement procedure with target bonded distributions obtained from all-atom simulations. The membrane thickness, area of the LPS, and density distributions for the different regions as well as the water and ion densities in Martini-3 simulations show excellent agreement with the all-atom data. Additionally the solvent accessible surface area for individual molecules in water was found to be in good agreement. The binding of calcium ions with phosphate and carboxylate moieties of LPS is accurately captured in the Martini-3 model, indicative of the integrity of the highly negatively charged LPS molecules in the outer membranes of Gram-negative bacteria. The melting transition of the coarse-grained lipid A membrane model was found to occur between 300 and 310 K, and the model captured variations in area per LPS, order parameter, and membrane thickness across the melting transition. Our study reveals that the proposed Martini-3 models for LPS are able to capture the physicochemical balance of the complex sugar architecture of the outer membrane of Escherichia coli. The coarse-grained models developed in this study would be useful for determining membrane protein interactions and permeation of potential antimicrobials through bacterial membranes at mesoscopic spatial and temporal scales.

Taxogenomics of Alkalihalobacterium chitinilyticum sp. nov.: an alkaliphilic chitin degrading bacterial strain isolated from Lonar Lake, India, with potential biotechnological applications.

A novel chitin degrading alkaliphilic bacterial strain (MEB 203T) was isolated from sediment collected from Lonar lake, India. The strain exhibited its maximum growth at a temperature of 37°C, with an optimal pH of 10 and a NaCl concentration of 2%. 16S rRNA gene based phylogenetic tree showed that strain was closely related to Alkalihalobacterium elongatum MCC 2982T (98.64% similarity) followed by A. alkalinitrilicum DSM 22532T (97.84% similarity). The genome size was 4.9Mb with DNA G + C content of 37.7%. The dDDH value between strain MEB 203T and A. elongatum MCC 2982T was 26.4 ± 2.4% while OrthoANI value was 82.1%. Genome analysis revealed the presence of genes responsible for L-ectoine and cation/proton antiporter which may facilitate growth of strain in alkaline-saline habitat of Lonar lake. Strain MEB 203T was able to utilize complex sugars such as chitin, cellulose, and starch as a carbon source at alkaline conditions which was also corroborated from the genomic presence of carbohydrate active enzymes (CAZymes). It was also able to produce biotechnologically important enzymes such as lipases and proteases which were stable at pH (9-10). The bacterium is majorly composed of C15:0 iso, C16:0 iso, and C17:0 iso (> 10%) fatty acids while diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and unidentified phospholipid (PL3) were identified as the predominant polar lipids. Based on differential physiological, biochemical, and genomic features of strain MEB 203T, a novel species Alkalihalobacterium chitinilyticum sp. nov. (Type strain MEB 203T = MCC 3920T = NCIMB 15407T = JCM 35078T) is proposed.

Complex organic matter degradation by secondary consumers in chemolithoautotrophy-based subsurface geothermal ecosystems.

Microbial communities in terrestrial geothermal systems often contain chemolithoautotrophs with well-characterized distributions and metabolic capabilities. However, the extent to which organic matter produced by these chemolithoautotrophs supports heterotrophs remains largely unknown. Here we compared the abundance and activity of peptidases and carbohydrate active enzymes (CAZymes) that are predicted to be extracellular identified in metagenomic assemblies from 63 springs in the Central American and the Andean convergent margin (Argentinian backarc of the Central Volcanic Zone), as well as the plume-influenced spreading center in Iceland. All assemblies contain two orders of magnitude more peptidases than CAZymes, suggesting that the microorganisms more often use proteins for their carbon and/or nitrogen acquisition instead of complex sugars. The CAZy families in highest abundance are GH23 and CBM50, and the most abundant peptidase families are M23 and C26, all four of which degrade peptidoglycan found in bacterial cells. This implies that the heterotrophic community relies on autochthonous dead cell biomass, rather than allochthonous plant matter, for organic material. Enzymes involved in the degradation of cyanobacterial- and algal-derived compounds are in lower abundance at every site, with volcanic sites having more enzymes degrading cyanobacterial compounds and non-volcanic sites having more enzymes degrading algal compounds. Activity assays showed that many of these enzyme classes are active in these samples. High temperature sites (> 80°C) had similar extracellular carbon-degrading enzymes regardless of their province, suggesting a less well-developed population of secondary consumers at these sites, possibly connected with the limited extent of the subsurface biosphere in these high temperature sites. We conclude that in < 80°C springs, chemolithoautotrophic production supports heterotrophs capable of degrading a wide range of organic compounds that do not vary by geological province, even though the taxonomic and respiratory repertoire of chemolithoautotrophs and heterotrophs differ greatly across these regions.

Description of Sporanaerobium hydrogeniformans gen. nov., sp. nov., an obligately anaerobic, hydrogen-producing bacterium isolated from Aravali hot spring in India.

An obligately anaerobic bacterium XHS1971T, capable of degrading cellulose and xylan, was isolated from a sediment sample of Aravali hot spring, Ratnagiri, India. Cells of strain XHS1971T were Gram-stain-negative, spore-forming, motile, long-rods. Growth was observed at temperatures 30-50°C (optimum 40-45°C), pH 5.0-10.0 (optimum pH 8.0) and NaCl concentrations 0-0.5% (optimum 0%). Generation time of strain XHS1971T was 5h under optimised growth conditions. Strain XHS1971T showed the ability to metabolise different complex and simple sugars constituting lignocellulosic biomass. Glucose was fermented majorly into hydrogen, formic acid, acetic acid, and ethanol, whereas carbon dioxide, butyric acid, lactic acid and succinic acid were produced in traces. 16S rRNA gene analysis of strain XHS1971T revealed < 94.5% homology with Cellulosilyticum lentocellum DSM5427T followed by Cellulosilyticum ruminicola JCM14822T, identifying strain as a distinct member of family Lachnospiraceae. The major cellular fatty acids (> 5%) were C14:0, C16:0, C18:0, and C16:1 ω7c. The genome size of the strain was 3.74Mb with 35.3mol% G + C content, and genes were annotated to carbohydrate metabolism, including genes involved in the degradation of cellulose and xylan and the production of hydrogen, ethanol and acetate. The uniqueness of strain was further validated by digital DNA-DNA hybridisation (dDDH), Average Nucleotide Identity (ANI), and Average Amino Acid Identity (AAI) values of 22%, 80%, and 63%, respectively, with nearest phylogenetic affiliates. Based on the detailed analyses, we propose a new genus and species, Sporanaerobium hydrogeniformans gen. nov., sp. nov., for strain XHS1971T (= MCC3498T = KCTC15729T = JCM32657T) within family Lachnospiraceae.

Glycoside hydrolases from (hyper)thermophilic archaea: structure, function, and applications.

(Hyper)thermophilic archaeal glycosidases are enzymes that catalyze the hydrolysis of glycosidic bonds to break down complex sugars and polysaccharides at high temperatures. These enzymes have an unique structure that allows them to remain stable and functional in extreme environments such as hot springs and hydrothermal vents. This review provides an overview of the current knowledge and milestones on the structures and functions of (hyper)thermophilic archaeal glycosidases and their potential applications in various fields. In particular, this review focuses on the structural characteristics of these enzymes and how these features relate to their catalytic activity by discussing different types of (hyper)thermophilic archaeal glycosidases, including β-glucosidases, chitinase, cellulases and α-amylases, describing their molecular structures, active sites, and mechanisms of action, including their role in the hydrolysis of carbohydrates. By providing a comprehensive overview of (hyper)thermophilic archaeal glycosidases, this review aims to stimulate further research into these fascinating enzymes.

Colonial Capitalist Heterochronicity: Socio-Ecological Rhythms of the Sugar Plantation and the Formal Subsumption of Historical and Cultural Difference

The Black and Third World Marxist tradition have demonstrated that colonialism is inseparable from historical accounts of global capitalism. This paper contributes to that project through an account of heterochronic capitalist time by indexing both its uneven incorporation of socio-ecological temporalities and its disciplining of enslaved people. To illustrate this, I examine how Western industrial temporal relations are generative of, and imposed through, its conflictual relations with Indigenous Taíno and enslaved West African socio-ecological forms of time within the Caribbean sugar complex. In addition, I emphasize that despite colonial capitalism seeking to merge African and Indigenous socio-ecological temporal knowledge into abstract labour, it is never a totalizing process. In effect, while colonial capitalism wields various techniques to incorporate Indigenous and African life worlds, there are always phenomenological remainders of cultural temporal difference that do not reproduce the logic of capital. Highlighting two contrasting postcolonial readings of Marx’s notion of subsumption, I argue that we can index the existence of a multiplicity of non-linear and cyclical forms of eternal time that comingle and link past, present and futurity. Inscribing their own emergent dialectics, however, I caution that preserved forms of temporal difference can potentially be taken up in service to reactionary political projects.

Integrated Sugar Industry Complex in India: Comparison of Different Potential Configurations

In this work, a multiproduct sugar industry complex with six different configurations is envisaged in the Indian context, and each of these configurations is evaluated using sustainability metrics. The product portfolio of the integrated sugar complex constitutes sugar, ethanol, and one of six possible bagasse valorization products, namely, electricity, Bio-CNG, 2G ethanol, lactic acid, succinic acid, and xylitol. Material and energy balance for the integrated complex is carried out by adapting process data from various reports, experiments, and simulation studies. A detailed economic analysis is performed for different capacities and operating days of the integrated complex, followed by cradle-to-gate LCA and a social assessment. The results showed that xylitol production for a sugar mill processing 325 tph was economically the most favorable with an NPV of US $86.48 million. Bio-CNG production was desirable from a climate change impact reduction standpoint. All configurations showed improvement over the business-as-usual scenario. Social assessment revealed that stakeholders displayed a strong inclination toward Bio-CNG production.

Stacy Malaker

The tattoo on Stacy Malaker’s ankle bears inky testament to her fascination with glycobiology. It depicts the core of a sugar molecule—a six-membered oxane ring, arranged in the classic chair configuration. “I figured I’ve devoted my entire career/life to glycobiology, so it made a little bit of sense,” she says. Despite the simple elegance of the tattoo, Malaker’s research involves more complex sugar chains called glycans. These unite with proteins to form glycoproteins such as mucins, which are major components of mucus and provide a protective barrier for tissues and organs. Malaker, an assistant professor of chemistry at Yale University, is developing mass spectrometry methods to analyze and understand mucins, which also play various roles in cancer and lung diseases. She hopes the research could eventually provide better ways to diagnose and monitor such conditions. “The area that she’s specializing in—mucin biology—is so important,” says Benjamin Garcia , a mass

Valorization of bioethanol by-products to produce unspecific peroxygenase with Agrocybe aegerita: Technological and proteomic perspectives

Unspecific peroxygenase (UPO) presents a wide range of biotechnological applications. This study targets the use of by-products from bioethanol synthesis to produce UPO by Agrocybe aegerita. Solid-state and submerged fermentations (SSF and SmF) were evaluated, achieving the highest titers of UPO and laccase in SmF using vinasse as nutrients source. Optimized UPO production of 331 U/L was achieved in 50% (v:v) vinasse with an inoculum grown for 14 days. These conditions were scaled-up to a 4 L reactor, achieving a UPO activity of 265 U/L. Fungal proteome expression was analyzed before and after UPO activity appeared by shotgun mass spectrometry proteomics. Laccase, dye-decolorizing peroxidases (DyP), lectins and proteins involved in reactive oxygen species (ROS) production and control were detected (in addition to UPO). Interestingly, the metabolism of complex sugars and nitrogen sources had a different activity at the beginning and end of the submerged fermentation.

Physiological Benefits of Dietary Lysophospholipid Supplementation in a Marine Fish Model: Deep Analyses of Modes of Action.

Given the hydrophilic structure of lysophospholipids (LPLs), their dietary inclusion translates into a better emulsifying capacity of the dietary components. The present study aimed to understand the mechanisms underlying the growth-promoting effect of LPL supplementation by undertaking deep analyses of the proximal intestine and liver interactomes. The Atlantic salmon (Salmo salar) was selected as the main aquaculture species model. The animals were divided into two groups: one was fed a control diet (C-diet) and the other a feed (LPL-diet) supplemented with an LPL-based digestive enhancer (0.1% AQUALYSO®, Adisseo). The LPL-diet had a positive effect on the fish by increasing the final weight by 5% and reducing total serum lipids, mainly due to a decrease in the plasma phospholipid (p < 0.05). In the intestine, the upregulated interactome suggests a more robust digestive capacity, improving vesicle-trafficking-related proteins, complex sugar hydrolysis, and lipid metabolism. In the liver, the LPL-diet promotes better nutrients, increasing several metabolic pathways. The downregulation of the responses to stress and stimuli could be related to a reduced proinflammatory state. This study on the benefits and modes of action of dietary LPLs opens a new window into fish nutrition and could be extended to other productive species.

SiaNAl can be efficiently incorporated in glycoproteins of human mesenchymal stromal cells by metabolic glycoengineering.

Metabolic glycoengineering involves the stimulation of cells with functionalized monosaccharides. Glucosamine, galactosamine, and mannosamine derivatives are commercially available, but their application may lead to undirected (i.e., chemical) incorporation into proteins. However, sialic acids are attached to the ends of complex sugar chains of glycoproteins, which might be beneficial for cell surface modification via click chemistry. Thus, we studied the incorporation of chemically synthesized unnatural alkyne modified sialic acid (SiaNAl) into glycoproteins of human telomerase-immortalized mesenchymal stromal cells (hMSC-TERT) and we show that SiaNAl can be efficiently incorporated in glycoproteins involved in signal transduction and cell junction.

Composition analysis of gastrointestinal contents of laboratory‐reared and wild lake sturgeon (Acipenser fulvescens)

AbstractThis study assessed the composition of digesta from captive and wild Acipenser fulvescens to consider how food availability and dietary absorption may influence stocking survival. Non‐lethal gastric lavage and colonic flushing were evaluated in laboratory fish and then in wild A. fulvescens caught from the Tennessee River. Gastrointestinal contents were collected at 2, 4, 6, 12, 24, and 32 h post‐feeding. The stomach content relative to ingested food was 36.67% ± 5.09% at 2 h post‐feeding, peaking at 4 h (75.82% ± 14.09%), and no food was recovered after 12 h. The colonic solid waste (g/kg) increased with time, with the highest recovery (5.40 ± 1.30 g/kg) at 32 h. Proximate composition of collected samples was determined using standard colorimetric assay protocols. There was a significant reduction in protein concentration in the stomach starting at 2 h, and glucose concentrations were stable in both stomach and colonic contents from 2 to 32 h post‐feeding in laboratory animals, suggesting longer processing times for dietary glucose compared to other nutrients. The protein concentration of recovered stomach solids was very similar between laboratory and wild populations (46.17 ± 7.32 vs. 40.00 ± 6.57 mg/dL) and in colonic solids (22.99 ± 4.26 vs. 19.86 ± 3.95 mg/dL). Lipid concentrations, however, deviated substantially, with 40.11 ± 7.71 mg/dL in stomach and 35.59 ± 7.29 mg/dL in faeces of laboratory fish, and only 18.26 ± 3.47 mg/dL (stomach) and 22.00 ± 5.54 mg/dL (colonic) in wild fish. Glucose represented a relatively small fraction in both stomach and colonic contents and differed based on habitat only in the colon, with wild fish retaining complex sugars for longer periods. These results indicate that high‐energy diet components are either reduced in natural diets or are preferentially absorbed within the first 12 h compared to laboratory fish. More study on use of lipid‐dense foods in the riverine environment may provide information to improve the survival of A. fulvescens to support their reintroduction.

Microbial Production of Human Milk Oligosaccharides.

Human milk oligosaccharides (HMOs) are complex nonnutritive sugars present in human milk. These sugars possess prebiotic, immunomodulatory, and antagonistic properties towards pathogens and therefore are important for the health and well-being of newborn babies. Lower prevalence of breastfeeding around the globe, rising popularity of nutraceuticals, and low availability of HMOs have inspired efforts to develop economically feasible and efficient industrial-scale production platforms for HMOs. Recent progress in synthetic biology and metabolic engineering tools has enabled microbial systems to be a production system of HMOs. In this regard, the model organism Escherichia coli has emerged as the preferred production platform. Herein, we summarize the remarkable progress in the microbial production of HMOs and discuss the challenges and future opportunities in unraveling the scope of production of complex HMOs. We focus on the microbial production of five HMOs that have been approved for their commercialization.

Tracking defined microbial communities by multicolor flow cytometry reveals tradeoffs between productivity and diversity.

Cross feeding between microbes is ubiquitous, but its impact on the diversity and productivity of microbial communities is incompletely understood. A reductionist approach using simple microbial communities has the potential to detect cross feeding interactions and their impact on ecosystem properties. However, quantifying abundance of more than two microbes in a community in a high throughput fashion requires rapid, inexpensive assays. Here, we show that multicolor flow cytometry combined with a machine learning-based classifier can rapidly quantify species abundances in simple, synthetic microbial communities. Our approach measures community structure over time and detects the exchange of metabolites in a four-member community of fluorescent Bacteroides species. Notably, we quantified species abundances in co-cultures and detected evidence of cooperation in polysaccharide processing and competition for monosaccharide utilization. We also observed that co-culturing on simple sugars, but not complex sugars, reduced microbial productivity, although less productive communities maintained higher community diversity. In summary, our multicolor flow cytometric approach presents an economical, tractable model system for microbial ecology using well-studied human bacteria. It can be extended to include additional species, evaluate more complex environments, and assay response of communities to a variety of disturbances.

Characterization and protein engineering of glycosyltransferases for the biosynthesis of diverse hepatoprotective cycloartane-type saponins in Astragalus membranaceus.

Although plant secondary metabolites are important source of new drugs, obtaining these compounds is challenging due to their high structural diversity and low abundance. The roots of Astragalus membranaceus are a popular herbal medicine worldwide. It contains a series of cycloartane-type saponins (astragalosides) as hepatoprotective and antivirus components. However, astragalosides exhibit complex sugar substitution patterns which hindered their purification and bioactivity investigation. In this work, glycosyltransferases (GT) from A. membranaceus were studied to synthesize structurally diverse astragalosides. Three new GTs, AmGT1/5 and AmGT9, were characterized as 3-O-glycosyltransferase and 25-O-glycosyltransferase of cycloastragenol respectively. AmGT1G146V/I variants were obtained as specific 3-O-xylosyltransferases by sequence alignment, molecular modelling and site-directed mutagenesis. A combinatorial synthesis system was established using AmGT1/5/9, AmGT1G146V/S and the reported AmGT8 and AmGT8A394F . The system allowed the synthesis of 13 astragalosides in Astragalus root with conversion rates from 22.6% to 98.7%, covering most of the sugar-substitution patterns for astragalosides. In addition, AmGT1 exhibited remarkable sugar donor promiscuity to use 10 different donors, and was used to synthesize three novel astragalosides and ginsenosides. Glycosylation remarkably improved the hepatoprotective and SARS-CoV-2 inhibition activities for triterpenoids. This is one of the first attempts to produce a series of herbal constituents via combinatorial synthesis. The results provided new biocatalytic tools for saponin biosynthesis.

The C-Type Lectin Receptor Dectin-2 Is a Receptor for Aspergillus fumigatus Galactomannan.

Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly among immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient serum and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the immune system and its consequences. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of highly purified Aspergillus galactomannan to identify a C-type lectin host receptor for this fungal carbohydrate. We identified a novel and specific interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2-dependent signaling, including activation of spleen tyrosine kinase, gene transcription, and tumor necrosis factor alpha (TNF-α) production. Deficiency of Dectin-2 increased immune cell recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for galactomannan, which leads to a proinflammatory immune response in the lung. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes serious and often fatal disease in humans. The surface of Aspergillus is composed of complex sugar molecules. Recognition of these carbohydrates by immune cells by carbohydrate lectin receptors can lead to clearance of the infection or, in some cases, benefit the fungus by dampening the host response. Galactomannan is a carbohydrate that is part of the cell surface of Aspergillus but is also released during infection and is found in patient lungs as well as their bloodstreams. The significance of our research is that we have identified Dectin-2 as a mammalian immune cell receptor that recognizes, binds, and signals in response to galactomannan. These results enhance our understanding of how this carbohydrate interacts with the immune system at the site of infection and will lead to broader understanding of how release of galactomannan by Aspergillus effects the immune response in infected patients.

Thermotoga neapolitana.

Like other Thermotogales, Thermotoga neapolitana produces a large quantity of hydrogen using proton as the final electron acceptor. It can utilize a wide range of simple and complex sugars, proteins, and peptides, as well as agricultural and food wastes as carbon and nitrogen sources. Its thermostable enzymes have been used in many industrial settings, such as food processing and consumer products. Biohydrogen gas can power fuel cells, releasing water as the only waste, an extremely appealing prospect in urban transportation.

Feed and biological value of the alfalfa green mass carbohydrate-lignin complex

Purpose. To study how the content of the main nutrients, the amount and ratio of structural and nonstructural carbohydrates in the green mass of alfalfa changes during the process of growth and development. To establish a phase in which the produced feed will have high feed and biological value. Methods. Sampling was carried out in accordance with the State Standard of Ukraine (SSU) ISO 6497:2005, determination of the content of moisture and other volatile substances – SSU ISO 6496:2005, nitrogen and crude protein – SSU 7169:2010, crude fat – SSU ISO 6492:2003, fiber – SSU 8844: 2019, raw ash – SSU ISO 5984:2004, carbohydrate-lignin complex (sugars, hemicellulose, cellulose) – SSU 7982:2015, neutral detergent fiber – SSU ISO 16472, acid detergent fiber and lignin – SSU ISO 13906:2013, starch – SSU ISO 6493:2008. Results. During alfalfa green mass growth and development, the content of dry matter increases gradually from 15% to 25%, the content of crude protein in it decreases from 26% to 13%, and crude fiber increases from 15% to 38%, the content and composition of nitrogen-free extractives change. In the phase of branching, the cellulose content is 12%, in the phase of full flowering – 24.6%, during beans development – 30.6%. The lignin content increases from 2.3% to 14.4%, that is, more than six times. The feeding value, expressed in the feed unit indicator, decreases from 0.9 to 0.79, and the exchangeable energy in the phase of budding and the beginning of flowering is at the level of 9.5 MJ. Conclusions. The green mass of alfalfa in the phase of budding-beginning of flowering is a valuable raw material for the production of feeds (hay, silage, granulated feed, grass flour, etc.), since the yield of the crop in this phase is still high, the crude protein content is at the 20% level, the amount of easily hydrolyzed carbohydrates is 14%, the amount of structural carbohydrates is still at the 30.0% level. The calculated feeding value of green mass is higher than the feeding value of the produced hay in terms of feed units by only 3.5%, digestible protein by 11%, and exchangeable energy by 2.6%.