BIOBUTANOL. PRODUCERS, SUBSTRATES, CULTIVATION AND RECOVERY
Authors:   Institute of Food Biotechnology and Genomics of the National Academy of Sciences of Ukraine, Shulga Serhiy , Tigunova Olena ,   Institute of Food Biotechnology and Genomics at the National Academy of Sciences of Ukraine, Bratishko Vyacheslav ,   Mechanical and Technologicalfaculty at the National University of Life and Environmental Sciences of Ukraine, Blume Yaroslav ,   Institute of Food Biotechnology and Genomics at the National Academy of Sciences of Ukraine
Published: May 2024

The rapid depletion of fossil fuel reserves and unpredictable fluctuations in crude oil prices due to geopolitical instability and rising energy demand have spurred research and development of various alternative fuels, especially biomass fuels. This shift has also been fuelled by environmental concerns arising fromgrowing greenhouse gas emissions and global warming, which have exacerbated the need for the most environmentally friendly type of fuel. In recent decades, several international projects focused on leveraging renewable energy sources for primary consumption have emerged. Notably, there has been a growing industrial focus on producing liquid biofuels like ethanol and butanol from specific renewable biomass sources such as agricultural residues, forest waste, or energy crops. While the term “biomass” has varied interpretations across different fields, Ukraine’s “Law on Alternative Fuels” provides a basic definition: it defines biomass as “a non-fossil biologically renewable organic substance, including products, waste, and residues from forestry, agriculture (plant and animal husbandry), fisheries, and related industries, as well as any industrial or household waste that can decompose biologically.”Biobutanol’s high energy density, low volatility, and potential for carbon neutrality make it a promising alternative to gasoline. It can be used as a fuel in road vehicles without any modifications to the internal combustion engine system. Many countries, including Ukraine, heavily rely on imported energy sources, driving a critical search for renewable alternatives. Over 50 countries now legally support developing renewable energy, switching to biofuels like biogas (produced from organic waste), bioethanol (e.g., from corn starch), biobutanol (from plant oils), and diesel biofuel, derived from various organic materials like agricultural and energy crops, forestry residues, and municipal waste. This transition is crucial for strengthening energy security, reducing carbon emissions, and promoting sustainable development. The monograph reflects the current state of development of various methods of biobutanol production, highlights the scientific basis of butanol production using microbiological synthesis in laboratory and industrial conditions, the history of its origin and development, the specifics and possibilities of its production, producers, substrates and equipment. Special attention in the monograph is paid to the history of industrial butanol production including recent advances in butanol production by ABE fermentation, highlighting its several new approaches. Thelatteris based on the search for inexhaustible and low-cost substrates and optimization of processes at the beginning and end of the technological chain. Since the production of butanol by ABE fermentation consists of several critical stages, the whole process in this monograph is described in five main phases: 1) selection of raw materials, 2) preliminary preparation of raw materials, 3) selection or creation of producers, 4) cultivation, 5) restoration of the final product The monograph also summarizes the latest achievements, as well as presents new challenges and potential prospects for research. The low yield of butanol and toxicity of the final product remain the major drawbacks of classical butanol production by ABE fermentation. The kind of raw material used as a substrate is an important factor in the production of biobutanol. Lignocellulosic biomass (e.g. agricultural, municipal waste and residues) can be used for the production of biobutanol but following a preliminary preparation. Specific feedstock pretreatment, fermentation strategies, bioreactor design and kinetics, and mathematical modelling can increase the efficiency of biobutanol production. For commercial industrial production, producers must be able to super-synthesize butanol. Modern genetic engineering methods of insertion, knockout and overexpression of genes to modify metabolic pathways can increase the production of biobutanol. Various genetic techniques, such as antisense RNA, TargeTron and CRISPR technology, have been used to generate efficient C.acetobutylicum producers in biobutanol production (Li et al., 2020). One of the approaches to continuous biomass bioconversion is the integration of the alcohol release stage with the fermentation process – the so-called extractive fermentation. At the first, acid production, stage of the ABA fermentation, C. acetobutylicum bacteria produce butyric, propionic, lactic and acetic acids.After that,when the level of hydrogen decreases, the synthesis of solvents – butanol, acetone, ethanol and isopropanol begins. This stage is initiated by an increase in the butyric acid concentration (more than 2 g/l) and a drop in the hydrogen index, pH < 5. Using traditional ABE fermentation, the yield of butanol from glucose is relatively low, at around 15%, and rarely exceeds 25% of the mass of sugars in the enzymatic medium. Butanol production is limited by the fact that at a butanol concentration of 1-2%, the reproduction of microorganisms is significantly blocked, which leads to cessation of the fermentation.For this reason, the concentration of butanol in the classic АВЕ process does not exceed 1.5% of the total volume at a productivity of 4.5 g/l/h and the accumulation of less than 25% of the mass of sugars, mainly glucose (Tigunova et al., 2013). For the successful production of biobutanol, it is necessary to consider several critical issues, such as the development of clostridial and non-clostridial producers, innovative recovery methods, and the search for integration with the ABE fermentation process. The monograph examines the metabolic pathways of butanol synthesis by microorganisms and their regulation, the most promising producer strains for industrial production and methods of increasing their productivity, including the modification of strains by genetic engineering methods, the conditions and selection of optimal technological parameters of cultivation and solvent recovery methods.The monograph provides examples of the use of organic wastes as raw materials for the production of biobutanol and the degree of their bioconversion. It also summarizes the results of the authors’ own research on obtaining new butanol-producing strains of the genus Clostridium, and their identification by classical physiological-morphological and genetic methods, followed by the study of characteristics and biological features. The possibility of using non-food raw materials as a substrate for cultivation is shown and a comparison of various methods of preliminary preparation of raw materials and their effect on the accumulation of butanol in the culture liquid is made. Lignocellulosic biomass is the most important raw material for second-generation biofuels, widely available in nature. Agricultural, forestry, and municipal waste is the main source of lignocellulosic biomass, and their use as raw materials for the production of chemicals is an ideal way to achieve a closed carbon economy. However, two major problems hinder this promising approach. With biomass hydrolysis releasing potentially inhibitory substances and forming a sugar mixture, pentoses/hexose co-utilization and inhibitor tolerance are the two key points for efficient fermentation of biomass hydrolysates. Pentoses (mainly xylose and arabinose) and hexoses (mainly glucose, fructose, galactose and mannose) coexist in biomass hydrolysates, and achieving efficient pentose/hexose co-utilization has been a challenge for a long time. Genetic engineering methods were used to overcome this problem. Inhibitors such as furfural, 5-hydroxymethylfurfural (HMF), formate, acetate and levulinate are always formed during biomass pretreatment. The mode of pretreatment has a significant effect on the type and amount of inhibitor formed. At low concentrations, these compounds are not harmful to Clostridiastrains but can inhibit other bacteria. The monograph discusses the main approaches to overcoming difficulties in the joint use of pentoses/hexoses and tolerance to inhibitors. The monograph describes the problems of storage of producer strains and defines the environment for the protection of microorganisms during freeze-drying, and an attempt is made to summarize various aspects of recent research and development, including the author’s research, in the field of efficient biobutanol production. The authors of the monograph have attempted to provide their own assessment of the achievements in the field to date, to propose detailed realistic goals for each stage with a view to a coordinated policy of research, development and demonstration (R&D), and to propose a way to strengthen the biofuel market and to more widely deploy research on renewable energy sources. Given the recent advances in biotechnology and bioprocessing, there is no doubt that the biobutanol synthesis technology has a basis for commercialization. The ability of renewable energy sources to meet global energy needs, albeit not completely, but to a large extent, has been sufficiently proven. Special attention is currently paid to the chances of commercialization of biofuels, which provides a sound assessment of various technical and economic aspects of pilot energy production in the future. Each of the main links of renewable energy production technologies is considered: the creation of genetically modified producers with increased resistance to solvents and metabolically modified strains capable of using a variety of carbon sources to produce more butanol rather than other byproducts; joint cultivation of clostridia, during which simultaneous saccharification and fermentation takes place, which allows creating technologies that, if successfully implemented in production, can lead to the emergence of an economically justified and expedient technology for obtaining butanol. The Future of Renewable Energy will be viewed as a critical and authoritative source for strategic planning of renewable energy development worldwide.The current state of energy production and the accompanying new directions of biological processes in the production of biofuels provide the industry with a unique perspective on solving existing or future scientific challenges and their possible solutions, as well as the opportunity to orient biotechnology to the implementation of the modern concept of establishing commercial production of commodity bioproducts.The commercial production of the latter soon will have a far-reaching effect in realizing the goal of sustainable transformation of renewable resources to meet the energy needs of mankind.