Optimization of Methanotrophic Growth and Production of Poly(3-Hydroxybutyrate) in a High-Throughput Microbioreactor System

Abstract

Production of poly(3-hydroxybutyrate) (P3HB) from methane has economic and environmental advantages over production by agricultural feedstock. Identification of high-productivity strains and optimal growth conditions is critical to efficient conversion of methane to polymer. Current culture conditions, including serum bottles, shake flasks, and agar plates, are labor-intensive and therefore insufficient for systematic screening and isolation. Gas chromatography, the standard method for analysis of P3HB content in bacterial biomass, is also incompatible with high-throughput screening. Growth in aerated microtiter plates coupled with a 96-well Nile red flow-cytometric assay creates an integrated microbioreactor system for high-throughput growth and analysis of P3HB-producing methanotrophic cultures, eliminating the need for individual manipulation of experimental replicates. This system was tested in practice to conduct medium optimization for P3HB production in pure cultures of Methylocystis parvus OBBP. Optimization gave insight into unexpected interactions: for example, low calcium concentrations significantly enhanced P3HB production under nitrogen-limited conditions. Optimization of calcium and copper concentrations in the growth medium increased final P3HB content from 18.1% to 49.4% and P3HB concentration from 0.69 g/liter to 3.43 g/liter while reducing doubling time from 10.6 h to 8.6 h. The ability to culture and analyze thousands of replicates with high mass transfer in completely mixed culture promises to streamline medium optimization and allow the detection and isolation of highly productive strains. Applications for this system are numerous, encompassing analysis of biofuels and other lipid inclusions, as well as analysis of heterotrophic and photosynthetic systems. P oly(3-hydroxy)butyrate (P3HB) is a biodegradable, biological alternative to recalcitrant petroleum-based polymers (1, 2). Commercial production of P3HB and related polyhydroxyalkanoate (PHA) copolymers currently relies on agricultural sugar substrates, with resulting high material costs, negative environmental impacts, and uncertainties related to the volatilities of agricultural markets (3). Alternative production methods are under study, including production in transgenic plants as well as production from alternative carbon sources, including volatile fatty acids, glycerol, methanol, and methane (4). Due to the abundance of low-cost methane worldwide, production of P3HB by methanotrophic bacteria could dramatically reduce substrate costs, allowing renewable, biodegradable polymers to compete more effectively with conventional plastics (5, 6).Commercial production of P3HB from methane and other alternative carbon sources is contingent upon continued gains in efficiency. Growth rates, P3HB content, and culture density together dictate capital, operational, and extraction costs. These values can be improved through a variety of approaches, including selection of wild-type strains and enrichments with desirable characteristics, optimizati...

Factors affecting the selection of PHB accumulating methanotrophs from waste activated sludge while utilizing ammonium as their nitrogen source

“…The PHB production process is mainly divided into two main phases; a growth phase for the bacteria where all essential nutrients are available in order to increase the biomass density followed by deficiency conditions to induce PHB accumulation as intracellular inclusions . The effect of many factors on both stages were studied including but not limited to the nitrogen source, copper, iron, phosphorus concentrations and methane to oxygen ratios , . These studies provided the effect of these parameters on enzymes activities, growth kinetics, stoichiometry and inhibitory levels for Type II methanotrophs during both phases.…”

“…7 The effect of many factors on both stages were studied including but not limited to the nitrogen source, copper, iron, phosphorus concentrations and methane to oxygen ratios. 12,13 These studies provided the effect of these parameters on enzymes activities, growth kinetics, stoichiometry and inhibitory levels for Type II methanotrophs during both phases.…”

The Opportunity for High-Performance Biomaterials from Methane

“…In the majority of cases, simple batch reactors (such as bottles, shake flasks, tanks, or fermenters) have been used, with either a static head or flow through of gas and under static, shaken or stirred conditions. On a micro scale, Sundstrom and Criddle used a high-throughput microbioreactor with aerated microtiter plates for rapid screening and isolation [ 77 ]. On a larger lab scale, pressurised bioreactors were used by Wendlandt et al [ 27 ] while Zúñiga et al [ 105 ] used a two-phase partitioning bioreactor, Rahnama et al explored the use of a bubble column and vertical loop reactor [ 75 ], and Pfluger et al adapted a laboratory scale fluidized bed reactor for growth of methanotrophs [ 67 ].…”

The effect of methane and odd-chain fatty acids on 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) synthesis by a Methylosinus-dominated mixed culture

“…Many technoeconomic studies have demonstrated that the use of methane for PHA production can significantly reduce raw material cost, minimize environmental impact, and make PHA more attractive in economic perspectives (Rostkowski et al 2012;Levett et al 2016;Koller et al 2017). To date, many methane-utilizing cultures capable of PHA production have been reported in literature (Pfluger et al 2011;Pieja et al 2011aPieja et al , b, 2012Stein et al 2011;Zuniga et al 2011;Belova et al 2013;Matsen et al 2013;Rostkowski et al 2013;Yang et al 2013;Myung et al 2015;Sundstrom and Criddle 2015;Criddle and Myung 2016;Myung et al 2016Myung et al , 2017. However, in order to make PHA production more competitive also in economic terms, open mixed microbial cultures can be offered as an alternative strategy for producing PHAs at lower costs (Broholm et al 1992;Beun et al 2002;Salehizadeh and Van Loosdrecht 2004;Dias et al 2006;Helm et al 2006Helm et al , 2008Albuquerque et al 2010;Arcos-Hernandez et al 2010;Colombo et al 2017;Luangthongkam et al 2019).…”