Development of Methane-Utilizing Mixed Cultures for the Production of Polyhydroxyalkanoates (PHAs) from Anaerobic Digester Sludge.

The fundamental components required for scaling up the production of biogas-based biopolymers can be provided through a single process, that is, anaerobic digestion (AD). In this research, the possibility of enriching methane-utilizing mixed cultures from the AD process was explored as well as their capability to accumulate polyhydroxyalkanoates (PHAs). For almost 70 days of operation in a fed-batch cyclic mode, the specific growth rate was 0.078 ± 0.005 h-1 and the biomass yield was 0.7 ± 0.08 mg-VSS/mg-CH4. Adjusting the nitrogen levels in AD centrate resulted in results comparable to those obtained with a synthetic medium. The enriched culture could accumulate up to 51 ± 2% PHB. On the other hand, when the culturing medium was supplemented with valeric acid, the enriched bacteria were able to produce polyhydroxybutyrate- co-valerate (PHBV) up to 52 ± 6% with an HV percentage of 33 ± 5%. Increasing the valeric acid concentration in the culturing medium above 100 mg/L decreased the overall amount of PHBV by 60%, whereas the number of HV units incorporated was not affected. Changing the methane-to-oxygen ratio (M/O) from 1:1 to 4:1 caused an almost 80% decline in PHB accumulation. In addition, M/O had a significant effect on the fraction composition of PHBV at different valeric acid concentrations.

Influence of biomass density and food to microorganisms ratio on the mixed culture type I methanotrophs enriched from activated sludge.

Methanotrophic based process can be the remedy to offset the wastewater treatment facilities increasing energy requirements due to methanotroph's unique ability to integrate methane assimilation with multiple biotechnological applications like biological nitrogen removal and methanol production. Regardless of the methanotrophic process end product, the challenge to maintain stable microbial growth in the methanotrophs cultivation bioreactor at higher cell densities is one of the major obstacles facing the process upscaling. Therefore, a series of consecutive batch tests were performed to attentively investigate the biomass density influence on type I methanotrophs bacterial growth. In addition, food to microorganisms (F/M), carbon to nitrogen (C/N) and nitrogen to microorganisms (N/M) ratio effect on the microbial activity was studied for the first time. It was clarified that the F/M ratio is the most influencing factor on the microbial growth at higher biomass densities rather than the biomass density increase, whereas C/N and N/M ratio change, while using nitrate as the nitrogen source, does not influence methanotrophs microbial growth. These study results would facilitate the scaling up of methanotrophic based biotechnology by identifying that F/M ratio as the key parameter that influences methanotrophs cultivation at high biomass densities.

Screening for Methane Utilizing Mixed Communities with High Polyhydroxybutyrate (PHB) Production Capacity Using Different Design Approaches.

With the adverse environmental ramifications of the use of petroleum-based plastic outweighing the challenges facing the industrialization of bioplastics, polyhydroxyalkanoate (PHA) biopolymer has gained broad interest in recent years. Thus, an efficient approach for maximizing polyhydroxybutyrate (PHB) polymer production in methanotrophic bacteria has been developed using the methane gas produced in the anaerobic digestion process in wastewater treatment plants (WWTPS) as a carbon substrate and an electron donor. A comparison study was conducted between two experimental setups using two different recycling strategies, namely new and conventional setups. The former setup aims to recycle PHB producers into the system after the PHB accumulation phase, while the latter recycles the biomass back into the system after the exponential phase of growth or the growth phase. The goal of this study was to compare both setups in terms of PHB production and other operational parameters such as growth rate, methane uptake rate, and biomass yield using two different nitrogen sources, namely nitrate and ammonia. The newly proposed setup is aimed at stimulating PHB accumulating type II methanotroph growth whilst enabling other PHB accumulators to grow simultaneously. The success of the proposed method was confirmed as it achieved highest recorded PHB accumulation percentages for a mixed culture community in both ammonia- and nitrate-enriched media of 59.4% and 54.3%, respectively, compared to 37.8% and 9.1% for the conventional setup. Finally, the sequencing of microbial samples showed a significant increase in the abundance of type II methanotrophs along with other PHB producers, confirming the success of the newly proposed technique in screening for PHB producers and achieving higher PHB accumulation.

Enhancement of the cultivation process conditions of mixed culture methanotrophic Proteobacteria phylum enriched from waste activated sludge as the first step for value added recovery process.

Methanotrophs are of great interest due to their distinguish ability of recovering value-added commodities such as methanol and lipids while mitigating methane. The enhancement of methanotrophs cultivation process conditions is a pivotal step to develop a feasible methanotrophic bioreactor. In this study, multiple batch tests have been performed to evaluate the aqueous growth medium elements including nitrogen, copper, and biomass density and the gaseous headspace composition influence on methanotrophs activity and the associated microbial community. It was found that increasing copper concentration to 20 µM and nitrate concentration to 40 mM result in higher growth rate and yield. In contrast, increasing the biomass density resulted in a declination in the growth rate and yield. The 16S rRNA gene sequencing shows that the used culture was dominated by type I Methylomonas genus at relative abundance of 60.9% compare to 1% of the enriched population for type II and type III methanotrophs. Thereafter, the specific nitrate uptake rate has been determined to be ranging from 0.05 to 0.62 mgN-NO3/mgTSS/day based on the cultures OD600. The attained results would facilitate the continuous cultivation of type I methanotrophs dominated culture as a first step for any methanotrophic based biotechnology.