Community structure evolution and enrichment of glycogen-accumulating organisms producing polyhydroxyalkanoates from fermented molasses

Abstract

An open mixed culture was enriched with glycogen-accumulating organisms (GAOs) by using a sequencing batch reactor and treating an agroindustrial waste (sugar cane molasses) under cyclic anaerobic-aerobic conditions. Over a 1-year operating period, the culture exhibited a very stable GAO phenotype with an average polyhydroxyalkanoate (PHA) content of 17% total suspended solids. However, the GAO microbial community evolved over the course of operation to a culture exhibiting unusual characteristics in producing PHAs comprised of short-chain-length monomers, namely, 3-hydroxybutyrate, 3-hydroxy-2-methylbutyrate, 3-hydroxyvalerate, and 3-hydroxy-2-methylvalerate, and also, up to 31 mol% of the medium-chain-length (MCL) monomer 3-hydroxyhexanoate (3HHx). Microbial community analysis by fluorescence in situ hybridization revealed a concurrent long-term drift in the GAO community balance, from mainly "Candidatus Competibacter phosphatis" to mainly Defluviicoccus vanus-related organisms. The production of 3HHx was confirmed by (13)C nuclear magnetic resonance (NMR) and appeared to be related to the increased presence of D. vanus-related GAOs. These results suggest a broadened spectrum of material, chemical, and mechanical properties that can be achieved for biopolymers produced by open mixed cultures from fermented waste. The increased spectrum of polymer properties brings a wider scope of potential applications.

FIG. 1.

(a) Typical pH and redox profiles. (b) PHA, VFA, glycogen, DOC, and ammonia profiles during a cycle of the SBR. l, liter.

FIG. 2.

Polymer yield on substrate and evolution of the PHA monomer proportions over reactor operation time.

FIG. 3.

Natural-abundance ¹³C-NMR spectra of the polymers extracted from cells collected from the SBR. The peaks were identified by comparing them to results in the literature (24, 25).

FIG. 4.

Proposed metabolic pathways for synthesis of the different monomers observed in the PHAs produced by GAOs in this study. ED, Entner-Doudoroff pathway; TCA, tricarboxylic acid cycle; CoA, coenzyme A; CoASH, reduced form of CoA; ACP, acyl carrier protein; ACPSH, reduced form of ACP; FADH₂, flavin adenine dinucleotide (reduced form).

FIG. 5.

Evolution of the PHAs produced per C-mol of total carbon consumed during the anaerobic phase, glycogen consumed per C-mol of VFA consumed during the anaerobic phase, and PHA content at the end of the anaerobic phase of each cycle study.

FIG. 6.

Morphologies of bacteria present in the GAO mixed culture. (a1, b1, and c1) Phase-contrast images. (a2) Overlay of results for EUBmix with those for the specific probe GAOmix. (b2) Overlay of results for EUBmix with those for the specific probe TFOmix. (c2) Overlay of results for EUBmix with those for the specific probe DFmix. Magnification, ×1,000. Results with for EUBmix are in green, and those for specific probes in red.