The development of microbial vaccines dedicated for the hydrolysis of the biomass in the anaerobic digestion

Biogas production is one of the most promising methods of producing renewable energy with the use and management of by-products of agriculture, industry and municipal waste in the anaerobic digestion process. Over the past decades, the biogas production technology has been focused on the development and optimization of systems characterized by a high rate of anaerobic digestion of energy crops, as well as solid agro-industrial wastes. The anaerobic digestion process consists of four stages: (i) hydrolysis (degradation) of high-molecular mass organic compounds to smaller monomers (proteins are degraded to amino acids, carbohydrates – to simple sugars, lipids to glycerin and fatty acids); (ii) acidogenesis, i.e. decomposition of the hydrolyzed substances to, among others, organic acids, alcohols, etc.; (iii) acetogenesis – oxidation of the produced organic acids to acetates and acetic acid, and (iv) methanogenesis, i.e. the degradation of acetates and acetic acid to methane and carbon dioxide. Therefore, the end products of anaerobic digestion of biomass are: biogas, consisting mainly of methane (30-70%) and carbon dioxide (30-70%), as well as sludge with a reduced amount of organic compounds. The key process, often limiting the speed of these biochemical changes, is hydrolysis. In order to facilitate effective biomass degradation, its pretreatment is often required. Biological pretreatment, which can be an alternative to the physical and chemical methods, is based on the activity of microorganisms, mainly bacteria and fungi, which produce hydrolytic enzymes (mainly cellulolytic, proteolytic and lipolytic). The biological pretreatment of the substrate may include: (i) the addition of specific microorganisms or consortia of hydrolytic microorganisms to the bioreactor (bioaugmentation) or (ii) supplementation of the system only with hydrolytic enzymes. Although the latter solution can improve the efficiency of the process, the activity of exogenous enzymes is usually affected by many different factors, including the type and variability of the substrate, incubation time, process configuration and physico-chemical conditions (e.g., temperature and pH). Bioaugmentation of the system also increases the enzymatic activity, as the enzymes are produced in situ by the microorganisms introduced to the system. The simultaneous production of complexes of various types of hydrolytic enzymes by live microorganisms allows for the application of this method in a potentially wide range of processes of utilization of different types of substrates (depending on the availability of various organic compounds in the bioreactor tank). 9 Among the main problems and limitations of the use of bioaugmentation are: low enzymatic activity of the microorganisms and sensitivity to changing stress conditions (including pH, temperature and the presence of heavy metals and antibiotics/pharmaceuticals). For this reason, the use of microbial consortia rather than singlestrain inocula is preferred, as different strains often vary in enzymatic properties or the range of tolerance to stress factors. This helps to maintain the activity of at least some members of the consortium over a wide range of conditions, on different types of substrates. Therefore, the most important step in the development of microbial vaccines is the selection and use of specialized microorganisms with a high biotechnological potential. In addition, the developed vaccines must meet the (bio)safety requirements for specific applications. The results obtained in this work increase the knowledge on the selection of microorganisms with cellulolytic, proteolytic and lipolytic activities, and constitute guidelines for the development of microbiological vaccines to be used for the degradation of organic compounds contained in plant biomass and sewage sludge, and increased biogas production. AIM The aims of the research were the following: (i) the selection of natural consortia of cellulolytic microorganisms and analysis of the impact of the source of microorganisms on the enzymatic activity of these consortia (ii) isolation and physiological characterization of pure cultures of cellulolytic bacteria and the development of a new (artificial) microbial consortium with a high cellulolytic activity, (iii) determination of the impact of the use of the developed consortia (both the natural and artificial) on the efficiency of lignocellulosic biomass hydrolysis and biogas production from it, (iv) determination of the total biotechnological (metabolic) potential and biosafety of the selected proteolytic and lipolytic strains in the context of their use for degradation of organic compounds contained in the sewage sludge, (v) demonstration of the impact of application of the proteolytic, lipolitic and cellulolytic strains on the efficiency of hydrolysis of organic compounds (proteins, lipids and carbohydrates) and biogas production from sewage sludge, (vi) analysis of the impact of the source of isolation of bacterial strains (sewage sludge, agricultural biogas plant) on their metabolic potential.