Single cell oil production: a new approach in biorefinery

A socially responsible economic growth, devoted to the future generations, requires long-term secure and available resources for industrial production, in terms of raw materials, energy and water. It should be environmentally friendly and a forward-looking financial system capable of future challenges with a global point of view. However, the present economic model based on the non-renewable fossil resources (oil, natural gas, coal, minerals) for energy and industrial production is the reason of energy instability, climate changes and therefore it cannot be considered sustainable. In this context, biotechnological techniques, such as the biorefinery are becoming more attractive. The biorefinery consist in the sustainable transformation of biomass, such as plant, algae, yeast, bacteria, into a wide range of marketable products and, in the mean time, into energy. Oils derived from biomass sources are named Microbial Oils, Unicellular Oils or Single Cell Oils (SCOs). The sustainable production of Single Cell Oils (SCOs) has garnered recent attention. The goal is new strategies or the biochemical/microbial conversion processes in order to increase their productivity and competitiveness. The identified strategies include the metabolic and genetic engineering of microorganisms, new fermentation technologies, innovative process choices, low-value feedstock and the recycle of by-products. SCOs are produced by oleaginous microorganisms which are able to accumulate between 20% and up to 80% lipid per dry biomass in the stationary growth phase under nutrient limitations, e.g., nitrogen or phosphorus, with simultaneous excess of carbon source. Depending on the oleaginous microorganisms including bacteria, yeasts, microalgae or fungal species, fatty acid profile of SCOs can vary making them highly suitable for different industrial applications. The SCOs, obtained from plant and microbial sources, offer several advantages, including faster production, less labor, more season and climate flexibility, and easier scale-up. The aim of this thesis is to explore the capacity, efficiency and productivity of oleaginous microorganisms, grown on agro-industrial lignocellulosic biomasses, to accumulate Single Cell Oils. This allow to reduce the environmental problem associated with the recycle of residues of various industrial processes allowing while increase the economic advantage linked to the SCO production. In order to make Single Cell Oil production more economical and sustainable, the experimental activities were aimed to achieve the following objectives: - The optimization of operating conditions of enzymatic hydrolysis evaluating the effects of enzyme concentration, temperature and pH on the fermentable sugar production. The experimental tests were carried out to assess the viability of innovative fermentation processes, i.e. in single stage or single reactor, with the aim to reduce the capital costs. It was found the synergism of enzymatic mix as well as the positive effects related to a lower temperature, suspended composition of hydrolysates, pH control and reaction time. - The implementation of enzymatic hydrolysis and oleaginous fermentation in single reactor (SRF) that offers an useful option to integrate in a single reactor two different stages of the microbial oil production: the enzymatic hydrolysis of the pretreated lignocellulosic biomass and the microbial fermentation of the obtained fermentable sugar mixture. Specific glucose consumption rate (μs), lipid yield (Ylipid) were calculated. The results suggest positive potential application of such process that still remains unexplored for Single Cell Oil production but demonstrate that they are suitable for biodiesel, bioplastic production or for other products of industrial interest. - The investigation of synergistic effect of yeast-microalga mixed cultures, in order to find suitable operating conditions to improve SCO production in mixotrophic microbial cultures. To test the consortium in clearly defined conditions, a synthetic medium was developed that integrates necessary elements of known culture media for both organisms, with the use of pH control. Growth series were done in batch, under constant light, agitation and temperature, and monitored gas exchange. In the model system, symbiotic growth was observed of the consortium with synergistic effects on biomass yield. Similar results were obtained in a system using lignocellulosic hydrolysate, except for an increase in lag phase due the presence of inhibitors. Growth even in anaerobic conditions (N2) confirmed synergistic interactions between the microalga and the oleaginous yeast. The performance of the consortium under different conditions is discussed in terms of growth rate, biomass production and lipid content of the biomass. - The implementation of yeast-microalga mixed cultures in open pond, overcoming contamination problems. The yeast grew, reaching the maximum concentration just after few days. The rapid growth kinetic was expected for the yeast while the very low cell proliferation can be explained by the low quantity of organic carbon in the medium

Biodiesel Production From Lignocellulosic Biomass Using Oleaginous Microbes: Prospects for Integrated Biofuel Production

Biodiesel is an eco-friendly, renewable, and potential liquid biofuel mitigating greenhouse gas emissions. Biodiesel has been produced initially from vegetable oils, non-edible oils, and waste oils. However, these feedstocks have several disadvantages such as requirement of land and labor and remain expensive. Similarly, in reference to waste oils, the feedstock content is succinct in supply and unable to meet the demand. Recent studies demonstrated utilization of lignocellulosic substrates for biodiesel production using oleaginous microorganisms. These microbes accumulate higher lipid content under stress conditions, whose lipid composition is similar to vegetable oils. In this paper, feedstocks used for biodiesel production such as vegetable oils, non-edible oils, oleaginous microalgae, fungi, yeast, and bacteria have been illustrated. Thereafter, steps enumerated in biodiesel production from lignocellulosic substrates through pretreatment, saccharification and oleaginous microbe-mediated fermentation, lipid extraction, transesterification, and purification of biodiesel are discussed. Besides, the importance of metabolic engineering in ensuring biofuels and biorefinery and a brief note on integration of liquid biofuels have been included that have significant importance in terms of circular economy aspects.Fil: Chintagunta, Anjani Devi. Vignan’s Foundation for Science, Technology and Research. Department of Biotechnology; IndiaFil: Zuccaro, Gaetano. Institut National de la Recherche Agronomique; Francia. Università degli Studi di Napoli Federico II; ItaliaFil: Kumar, Mahesh. Central Agricultural University; IndiaFil: Kumar, S. P. Jeevan. Indian Institute of Seed Science; India. Directorate of Floricultural Research; IndiaFil: Garlapati, Vijay Kumar. Jaypee University of Information Technology; IndiaFil: Postemsky, Pablo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; ArgentinaFil: Kumar, N. S. Sampath. Vignan’s Foundation for Science, Technology and Research. Department of Biotechnology; IndiaFil: Chandel, Anuj K.. Universidade de Sao Paulo; BrasilFil: Simal Gandara, Jesus. Universidad de Vigo; Españ

Biotechnological synthesis of succinic acid by actinobacillus succinogenes by exploitation of lignocellulosic biomass

Succinic acid is increasingly used in pharmaceutical industries, for the production of additives in food industries, in agriculture and in refinery processes as a precursor of many chemical compounds among which the most important is the succinate salt. It is also used as an ion chelator and surfactant, and for the biochemicals production. Currently, succinic acid is mainly produced through chemical petroleum-based processes, usually from n-butane using maleic anhydride. However, the use of petrochemical feedstocks raises serious environmental problems, due to the higher values of temperature and pressure required. The biotechnological production of succinic acid by microbial conversion of lignocellulosic biomass is attracting growing interest due to the environmental and economic advantages offered. This research is focused on the exploitation of Arundo donax (Giant reed) as a source of lignocellulosic biomass. Arundo donax is a perennial crop particularly suitable for energy production, as it offers high yields per hectare, even in partially fertile or polluted soils, not used for agriculture. Hydrolyzate of Arundo donax will be used as growth media for the Actinobacillus succinogenes 130Z, a bacterium typically found in the bovine rumen, that is recognized as one of the most promising for the biotechnological production of succinic acid, as it is able to produce higher concentrations of succinic acid. The experimental analysis is carried out to optimize the production of succinic acid taking into account the effect of the most critical parameters of the process (microbial biomass, pH, reducing sugars, volatile fatty acids, and succinic acid). Tests have shown that in 48h the sugars are completely biodegraded with a total production of bio-succinic acid of 5.9 g for 9.1 g of reducing sugars, an hourly production 0.12 g h-1 with a yield equal to 65%

Biochar addition in the anaerobic digestion of the organic fraction of municipal solid waste for biogas production

The continuous decline of fossil fuel availability and the ever increasing concern about environmental pollution, expressed by scientists, governments and public at large, are stimulating the research on renewable energy production. In this perspective, anaerobic digestion (AD) of the organic fraction of municipal solid waste (OFMSW) is recently meeting with increasing interest. It is a process viable both from an economic and technological standpoints, capable to combine the environmental friendly re-cycle of large amount of OFMSW combined to the production of methane, an excellent fossil-based fuels substitute (Chatterjee and Mazumder, 2016). Please click on the file below for full content of the abstract

Why We Should Support Biofuel Production

We are currently in a dynamic phase of civilisation, in which the technological progress that has drastically altered our lives is accompanied by other historical events that forcibly affect and will affect future choices [...

Lipid Production from Arundo Donax Grown under Different Agronomical Conditions

Hydrolysates of Giant reed (Arundo donax) biomass from three different agronomical conditions were used to grow the oleaginous yeast L. Starkey. The agronomical conditions affected the cellulose fraction of biomass, the amount of inhibitors generated during the acid hydrolysis, and the triglyceride yield after the yeast fermentation. Yet, the composition of triglycerides was not affected. Different approaches were developed to reduce the effect of inhibitors. The preliminary dilution of hydrolysates was studied, obtaining the highest values of biomass and lipid yields with a 50% dilution. Alternatively, the hydrolysates were pre-treated by adsorption and overliming. The latter pre-treatment gave the best results. A third approach was offered by the use of pre-adapted yeasts, that were able to grow in the presence of raw hydrolysates. The composition of the microbial triglycerides was compatible with the production of a biodiesel suitable for use as automotive fuel

Effect of Nickel Contamination on the Growth of Oleaginous Yeasts in Hydrolisates of Arundo Donax

Hydrolysates of Arundo donax, a crop offering high productivity in contaminated orsalinized soils with no inputs of irrigation and agrochemicals, were used in a discontinuous fermenter to grow the oleaginous yeast Lipomyces starkey, to obtain microbial oils potentially useful for the production of 2nd-generation biodiesel. A mixture of fermentable sugars was obtained by steam-explosion and subsequent enzymatic hydrolysis of the lignocellulosic materials. The concentration of Ni2+ ions and of inhibitors of the microbial growth significantly affected both the biomass and the triglyceride yields. The microbial lipids produced were compatible with the synthesis of an automotive-grade biodiesel. A physico-mathematical model, developed todescribe the biomass growth, demonstratedthat the concentration of heavy metals affected the maximum biomass concentration,though its influence on the specific growthrate of the yeasts was not significant

Effect of Nickel Contamination on the Growth of Oleaginous Yeasts in Hydrolisates of Arundo Donax

Hydrolysates of Arundo donax, a crop offering high productivity in contaminated orsalinized soils with no inputs of irrigation and agrochemicals, were used in a discontinuous fermenter to grow the oleaginous yeast Lipomyces starkey, to obtain microbial oils potentially useful for the production of 2nd-generation biodiesel. A mixture of fermentable sugars was obtained by steam-explosion and subsequent enzymatic hydrolysis of the lignocellulosic materials. The concentration of Ni2+ ions and of inhibitors of the microbial growth significantly affected both the biomass and the triglyceride yields. The microbial lipids produced were compatible with the synthesis of an automotive-grade biodiesel. A physico-mathematical model, developed todescribe the biomass growth, demonstratedthat the concentration of heavy metals affected the maximum biomass concentration,though its influence on the specific growthrate of the yeasts was not significant

Dark Fermentation of <i>Arundo donax:</i> Characterization of the Anaerobic Microbial Consortium

The dark fermentation of lignocellulose hydrolysates is a promising process for the production of hydrogen from renewable sources. Nevertheless, hydrogen yields are often lower than those obtained from other carbohydrate sources due to the presence of microbial growth inhibitors in lignocellulose hydrolysates. In this study, a microbial consortium for the production of hydrogen by dark fermentation has been obtained from a wild methanogenic sludge by means of thermal treatments. The consortium has been initially acclimated to a glucose-based medium and then used as inoculum for the fermentation of Arundo donax hydrolysates. Hydrogen yields obtained from fermentation of A. donax hydrolysates were lower than those obtained from glucose fermentation using the same inoculum (0.30 ± 0.05 versus 1.11 ± 0.06 mol of H2 per mol of glucose equivalents). The hydrogen-producing bacteria belonged mainly to the Enterobacteriaceae family in cultures growing on glucose and to Clostridium in those growing on A. donax hydrolysate. In the latter cultures, Lactobacillus outcompeted Enterobacteriaceae, although Clostridium also increased. Lactobacillus outgrowth could account for the lower yields observed in cultures growing on A. donax hydrolysate