Energetic utilization of lignocellulose-rich agricultural wastes by enriched microorganisms from high performance natural and engineered systems

Energy crops, agricultural residues, animal manure, forestry residues, algae, etc. are one of the major source for renewable energy production. The amount of lignocellulose-rich agricultural wastes (such as wheat straw, rice straw, etc.) increasing day by day due to the rapid growth on population which increased the pressure on agricultural industry to meet the demands for food and animal feed. In most of the countries, these wastes are handled in an unsustainable manner, mostly disposed by burning which also has a negative impact on global warming. On the other hand, these waste streams have a great potential to be converted into wide variety of value-added products including biofuels. Anaerobic digestion is a commonly used proven technology to recovery energy from various feedstocks including agricultural residues, livestock manure, food and brewery wastes, sewage sludge, industrial and municipal wastewaters, wood industry residues etc. Among these waste streams, agricultural residues belong to the most attractive substrates for anaerobic digesters due to the high abundance, high energy content and low costs. Anaerobic digestion is a complex biological process carried out by a diverse microbial community including hydrolytic, acidogenic and acetogenic bacteria and methanogenic archaea. During the process, organic content of the biomass is degraded by a synergistic interactions of the microbial community and converted into methane-rich biogas, then methane is used for energy production. One of the main obstacles during anaerobic digestion of lignocellulose-rich biomass is the recalcitrant structure which limits the efficiency of anaerobic digesters. Hydrolysis of these compounds is considered as the rate-limiting step. Investigating the efficient approach to overcome this problem is a continuing concern. Thus, different strategies such as physical, chemical and biological pre-treatment methods are applied to enhance hydrolysis and then the overall system performance, accordingly. As the diverse group of microorganisms are necessary to carry out anaerobic digestion process, the effective inoculum selection is crutial at the start-up period. Usually, anaerobic digesters are seeded by inoculum taken from another digester or animal manure which are easy to procure. However, the specific inocula should be intoduced to the specific feedstock to enhance start-up and acclimation periods, and methane yields. The nature is always an inspiration for technological developments. Biomimicry is an approach which interests engineers for many years for new innovations. Ruminants have a sophisticated digestive system which are able to degrade a wide variety of lignocellulosic substrates thanks to a symbiotic relationships in their digestive systems. Moreover, the microorganisms in the rumen fluid is more effective in lignocellulose digestion compared to the microorganisms in anaerobic digesters. However, procurement of the rumen fluid in necessary amounts to seed a full-scale anaerobic digester is not feasible. The selective enrichment of lignocellulose-degrading bacterial culture can be a solution to enhance methane yield in anaerobic digesters. This thesis intends to obtain the most effective bioaugmentation culture with an inspiration from the high performance natural and engineering systems to enhance the anaerobic digestion of lignocellulosic feedstock and determine the most effective concentration of the bioaugmentation cultures applied to anaerobic digesters. For this purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from rumen fluids of different ruminants (sheep, cow and goat) as well as a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate) under anaerobic mesophilic conditions. The bacterial communities were examined by 454 amplicon sequencing of 16S rRNA genes, while terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of mcrA genes was applied to analyze the methanogenic communities. The comparison of the enhancement potential of these bioaugmentation cultures were carried out in batch reactors using wheat straw as a lignocellulosic feedstock under mesophilic conditions. To investigate the effects of most effective concentration, the enrichment cultures were added to batch reactors in proportions of 2%, 4%, 6% and 8% of the microbial cell number of the standard inoculum slurry. All cultures had positive effects on substrate degradation, and higher methane yields were observed in the bioaugmented reactors compared to control reactors set up with standard inoculum. However, the level of enhancement differed according to the type of the enrichment culture. Whereas, addition of 2% sheep rumen derived enrichment culture did not enhance methane production significantly, 4% addition led an increase by 27%. Methane yield in batch reactors augmented with 2% cow rumen derived enrichment culture was increased by only 6%. In contrast, reactors amended with 2% goat rumen derived enrichment culture or with the bioaugmentation culture obtained from the biogas reactor digesting sorghum biomass produced 27% and 20% more methane, respectively. The highest methane yield was recorded in reactors amended with 6% goat rumen derived enrichment culture, which represented an increase by 36%. The results highlighted that relative abundances of Ruminococcaceae and Lachnospiraceae increased during the enrichment of sheep rumen fluid derived culture. On the other hand Bacteroidaceae and Ruminococcaceae were the most abundant families in the cow and goat rumen-derived enrichment cultures, Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure. Whereas, the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures derived from goat and cow rumen fluid as well as biogas reactor, the sheep rumen culture was dominated by Methanosarcinales. The microbial communities were quite similar at the end of the batch tests independently of the bioaugmentation sources, indicating that the introduced microbial communities of the enrichment cultures did not dominate the reactors. The results suggest that bioaugmentation with enrichment cultures derived from high performance natural and engineering systems can be an alternative strategy to enhance digester performance treating lignocellulosic feedstocks.