Cyclic, alternating methane and nitrogen limitation increases PHB production in a methanotrophic community Allison J. Pieja ⇑ , Eric R. Sundstrom, Craig S. Criddle Environmental Engineering and Science, Department of Civil and Environmental Engineering, Stanford University, Stanford, CA 94305-4020, USA article info Article history: Received 13 October 2011 Received in revised form 8 December 2011 Accepted 8 December 2011 Available online 27 December 2011 Keywords: Methanotroph PHB Bioreactor Feast–famine Selection abstract To identify feast–famine strategies that favor PHB accumulation in Type II methanotrophic proteobacte- ria, three sequencing batch reactors seeded with a defined inoculum of Type II methanotrophs were subjected to 24-h cycles consisting of (1) repeated nitrogen limitation, (2) repeated nitrogen and oxygen limitation, and (3) repeated nitrogen and methane limitation. PHB levels within each reactor and capacity to produce PHB in offline batch incubations were monitored over 11 cycles. PHB content increased only in the reactor limited by both nitrogen and methane. This reactor became dominated by Methylocystis par- vus OBBP with no detectable minority populations. It was concluded that repeated nitrogen and methane limitations favored PHB accumulation in strain OBBP and provided it with a competitive advantage under the conditions imposed. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Poly-3-hydroxyalkanoates (PHA) are biologically produced, bio- degradable polyesters with a chemical structure similar to poly- propylene and mechanical properties that can be tailored for different applications by changing the copolymer content of the polymer (Anderson and Dawes, 1990; Braunegg et al., 1998; Lee, 1996). Many bacteria accumulate PHA as a carbon storage polymer under conditions of unbalanced growth (i.e. nutrient deficiency and/or carbon excess) (Anderson and Dawes, 1990; Lee, 1996). De- spite considerable efforts, PHA commercialization has been limited because production costs are relatively high compared to those of traditional petrochemical-based plastics such as polyethylene and polypropylene (Lee et al., 2005). Approximately 30% of PHA production cost is attributed to feedstock (Choi and Lee, 1997), which is typically sugar (Lee, 1996). Use of waste-derived organics as feedstock can potentially reduce the cost of PHA production (Du et al., 2004; Braunegg et al., 1998; Koller et al., 2005; Ashby et al., 2004; Atlic et al., 2011). Methane is a potential low-cost, waste-derived feedstock for bioplastic production (Wendlandt et al., 2001; Pfluger et al., 2011). Methanotrophic cultures utilizing methane produce poly- 3-hydroxybutyrate (PHB), a specific PHA homopolymer, and have been shown to produce up to 51% PHB at a concentration of 30 g PHB/L in 24 h (Wendlandt et al., 2001). One way to increase the PHB production capacity of methano- trophic cultures and maintain a system under non-sterile condi- tions may be to operate a reactor under cyclical selection pressures that favor PHB accumulation. Repeated cycles of feast and famine (i.e. pulse feeding of acetate) increased both the PHA storage capacity and the PHA storage rate of non-sterile, mixed cul- tures (Dionisi et al., 2004; Johnson et al., 2009; Serafim et al., 2004). Acetate-fed cultures operated under a feast–famine regime store a significant fraction of carbon during the feast phase (Dircks et al., 2001). Theoretically, carbon storage provide a competitive advan- tage: during periods of carbon famine, stored PHA reserves can be used for replication, but microorganisms lacking such reserves cannot replicate and must survive without a carbon or energy source (Johnson et al., 2009). While a selection strategy based on feast–famine cycles for car- bon effectively enriched a community of acetate-fed microorgan- isms capable of rapidly producing PHA at up to 89% of the dry cell weight (Johnson et al., 2009), it is doubtful that such a strategy would succeed with methanotrophic bacteria. For Methylocystis parvus OBBP, methane can serve as a source of carbon and elec- trons, but PHB appears to serve only as a source of electrons that may be used for methane oxidation or for carbon or nitrogen assimilation (Chu and Alvarez-Cohen, 1998; Fitch et al., 1996; Henrysson and McCarty, 1993; Pieja et al., 2011b; Sipkema et al., 2000). Short-term methane starvation does not induce PHB con- sumption or replication in methanotrophs (Pieja et al., 2011b). It is, therefore, likely that a feast–famine approach, or pulse-feeding of methane, would not induce significant PHB production or consumption. 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.12.044 ⇑ Corresponding author. Tel.: +1 908 403 9630; fax: +1 650 725 3164. E-mail address: apieja@alumni.princeton.edu (A.J. Pieja). Bioresource Technology 107 (2012) 385–392 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech