Influence of the C/N ratio on the performance of polyhydroxybutyrate (PHB) producing sequencing batch reactors at short SRTs

Abstract

Many waste streams that are suitable substrates for mixed culture bioplastic (polyhydroxyalkanoate, PHA) production are nutrient limited and may need to be supplemented to allow sufficient growth of PHA accumulating bacteria. The scope of this study was to investigate the necessity of nutrient supplementation for the enrichment of an efficient PHA producing mixed culture. We studied the influence of different degrees of carbon and nitrogen limitation on the performance of an acetate-fed feast–famine sequencing batch reactor (SBR) employed to enrich PHA storing bacteria. The microbial reaction rates in the SBR showed a shift with a change in the limiting substrate: high acetate uptake rates were found in carbon-limited SBRs (medium C/N ratios 6–13.2 Cmol/Nmol), while nitrogen-limited SBRs (medium C/N ratios 15–24 Cmol/Nmol) were characterized by high ammonia uptake rates. Biomass in strongly nitrogen-limited SBRs had higher baseline PHA contents in the SBR, but carbon-limited SBRs resulted usually in biomass with higher maximal PHA storage capacities. The PHA storage capacity in a nitrogen-limited SBR operated at 0.5 d SRT decreased significantly over less than 5 months operation. For the microbial selection and biomass production stage of a PHA production process carbon limitation seems thus favourable and nutrient deficient wastewaters may consequently require supplementation with nutrients for the selection of a stable PHA storing biomass with a high storage capacity.

Introduction

Polyhydroxyalkanoates (PHA) are microbial storage polymers which increasingly attract interest as bioplastics. Industrial processes for the production of PHA are currently based on pure cultures of either genetically modified bacteria (e.g. Metabolix, U.S.A) or wild type PHA producing bacteria (e.g. Tianan Biological Materials, China). Process optimization, a growing competition on the PHA market, and larger scale production facilities have helped reducing the price of PHA to about 4.90 $/kg (Lunt, 2008), however, this price is still significantly higher than the price of conventional plastics such as polypropylene. As costs for raw materials play a crucial role for the economy of the PHA production process, alternative substrates derived from waste streams could help reduce the costs (Braunegg et al., 2004). The use of a PHA production process based on open mixed microbial cultures rather than on pure cultures would be favourable when using waste streams, since mixed microbial cultures can better adapt to changes in substrate supply and sterilization of the substrate, which can have detrimental effects on the substrate quality, would not be required. Industrial wastewaters suitable for mixed culture PHA production such as paper mill wastewater or molasses are often nutrient deficient. If nutrient supplementation would be required for these streams, this could be an important cost factor. The aim of this study was to investigate the impact of nutrient limitation as opposed to carbon limitation on the enrichment of a stable and efficient PHA producing mixed culture, in order to judge whether nutrient supplementation would be necessary for deficient waste streams.

Typically, the upstream part of a mixed culture PHA production process from waste streams would consist of three steps: (i) feedstock production through anaerobic mixed culture fermentation of waste streams to volatile fatty acids, (ii) culture selection, and (iii) PHA production (Serafim et al., 2008, van Loosdrecht, 2000). The second step makes use of the ecological role of PHA as a storage polymer in order to enrich a mixed culture capable of producing high amounts of PHA (van Loosdrecht et al., 1997). A successful strategy to enrich PHA producers in a mixed culture is the use of a dynamic feeding pattern consisting of alternating periods of presence (feast) and absence (famine) of the carbon source under fully aerobic conditions (also called aerobic dynamic feeding or feast–famine). Since this step is not only an enrichment step, but also the step in which the biomass is produced for the third step, nutrients and a carbon source are required for the biomass to proliferate. In the third step, the PHA production, biomass harvested from the second step is supplied with an excess of carbon source under conditions of nutrient limitation in order to minimize growth and maximize the cellular PHA content.

While the benefit of nutrient limitation on the maximum PHA content reached during the PHA production step has been documented (Bengtsson et al., 2008, Serafim et al., 2004), the optimal composition of the feed in terms of nutrient versus carbon source during the culture selection step remains unclear as indicated by the following examples.

Using nutrient deficient waste streams such as fermented molasses (Albuquerque et al., 2007), fermented paper mill effluent (Bengtsson et al., 2008) and fermented olive oil mill effluent (Beccari et al., 2009), PHA producing mixed cultures were successfully enriched, but in all studies the fermented substrate streams had to be supplemented with nutrients. Albuquerque et al. (2007) found that if the supplementation with a nitrogen source was not sufficient (nitrogen limitation), no stable PHA storing culture could be obtained. On the other hand other studies with cultures grown on acetate or propionate did show that stable PHA producing cultures with fairly high PHA storage capacities can be enriched with nutrient limited feeds (Lemos et al., 2006, Serafim et al., 2004). The influence of carbon versus nutrient limitation on the enrichment of a PHA producing culture requires thus further investigation.

We investigated the influence of the carbon to nitrogen ratio (C/N ratio) of the medium on the performance of a mixed culture in a selection reactor and on the culture's PHA production capacity. We performed culture selection experiments in sequencing batch reactors (SBR) at a range of C/N ratios from carbon to nitrogen-limited with acetate as the substrate. When a stable reactor operation was obtained the reactor behaviour was documented by measurements of the relevant state variables. The selected cultures were subsequently transferred into nutrient limited fed-batch reactors in order to evaluate the capacity of the selected cultures to store PHA. The PHA produced from acetate was pure polyhydroxybutyrate (PHB).

As it can be expected that also the sludge residence time (SRT) in the SBR has an influence on whether a particular culture will be carbon or nitrogen-limited at a certain C/N ratio, we also investigated cultures with different SRTs and the same medium C/N ratio.