Effect of dark/light periods on the polyhydroxyalkanoate production of a photosynthetic mixed culture
Introduction
In the last decades, we have witnessed an increasing demand for environmentally friendly materials that could replace the non-degradable plastics chemically synthesized from finite oil reserves. To address this demand, researchers have focused their attention on polyhydroxyalkanoates (PHAs), which are natural biodegradable polyesters internally synthesized by some microorganisms as carbon reserves. These biopolymers present thermoplastic properties similar to conventional plastics, making them potential replacement materials for petrochemically based plastics (Braunegg et al., 1998, Verlinden et al., 2007).
To date, PHA is already industrially produced using pure cultures and refined substrates under sterile operating conditions. However, in order to compete with the low price of traditional plastics, the current high costs of industrial PHA production must decrease (Reis et al., 2011). To make this biopolymer a more economically accessible material, several studies using mixed microbial cultures (MMC) fed with low/zero cost substrates have been proposed. Different feedstocks like sugar cane molasses (Albuquerque et al., 2010, Bengtsson et al., 2010), olive oil mill pomace (Waller et al., 2012), mixtures of sludge and food waste (Chen et al., 2013), and wastewater from paper mill effluents (Bengtsson et al., 2008, Jiang et al., 2012) have been used as substrates for PHA production and demonstrated comparable yields to PHA production with pure cultures, thus contributing to a decrease in PHA production costs while simultaneously treating polluted streams.
The selection of a MMC with a high PHA storing capacity using such agro-industrial wastes or by-products is usually performed through transient feeding conditions of the desired feedstock, in a strategy designated as Feast and Famine (FF). By feeding the culture intermittently, organisms that accumulate the substrate as PHA during the feed phase will be favoured during the famine phase, since only the organisms containing storage products will be able to grow in the absence of external substrate, thus enriching the MMC in PHA accumulating organisms (Reis et al., 2003). Results from the application of this strategy have been promising so far, high polymer content has been achieved, reaching maximum values of 77% PHA content when feeding paper mill wastewater (Jiang et al., 2012). Moreover, diverse monomeric compositions have been obtained, with co-polymers of short and medium chain length monomers being simultaneously produced, conferring broader thermal and mechanical properties to PHA (Pisco et al., 2009, Albuquerque et al., 2011).
Despite the efforts to develop less expensive PHA production systems, all of these studies utilizing mixed microbial cultures require intensive aeration, which substantially increases operational costs (Rosso et al., 2008). To avoid these aeration costs, a new MMC PHA production system has been recently proposed using photosynthetic microorganisms (Fradinho et al., 2013). In this photosynthetic system, the capability of microorganisms to accumulate PHA was explored, with the advantage that these photosynthetic organisms can obtain energy from light, thus, not requiring aeration for ATP production. Using a FF strategy and with constant illumination, Fradinho et al. (2013) enriched a photosynthetic mixed culture (PMC) composed of a consortium of bacteria and algae, with bacteria accumulating PHA in the feast phase, and consuming it in the famine phase using the oxygen produced by the algae. The PMC accumulated up to 20% PHA/VSS with acetate as the carbon source, with a yield of PHA per substrate of 0.70 Cmol PHA/Cmol Acet, which is similar to the yields obtained with aerobic MMC selected under feast/famine conditions.
Despite the elimination of aeration costs, the constant illumination of the proposed photosynthetic system would also add associated costs. One way of minimising the operational costs of the PMC in future applications would be the use of solar light as the illumination source. For this reason, it is critical to assess the impact of alternating dark/light periods on the performance of the PMC, thus evaluating if the photosynthetic culture can endure dark periods as would occur with direct sunlight utilization. In a batch test, Fradinho et al. (2013) tested the PMC behaviour in response to dark anaerobic conditions, and observed that internally stored carbohydrates were utilised as an energy source for acetate uptake, resulting in PHA production. However, long-term studies with sequential dark/light cycles are needed in order to determine if this capacity can be exploited to operate a PMC with effective PHA producing characteristics with reduced illumination periods. Therefore, in this work, we evaluate the possibility of enriching a viable photosynthetic mixed PHA accumulating culture in a regime with dark/light periods, and assess the impact of the illumination pattern on the culture community and its PHA accumulating capacity. The effect of this operational strategy was evaluated through assessing the stoichiometric and kinetic parameters of the PMC, which was accompanied by microscopic examination of the culture.
Section snippets
Mixed photosynthetic culture operation under dark/light cycles
The inoculum for the PMC studied in this work was obtained from a photosynthetic consortium of bacteria and algae that was operated with continuous illumination (1.3 W/L of culture broth) in a feast and famine regime, using acetate as carbon source (6 C-mM in the beginning of the feast phase) and containing a biomass concentration of 2.7 g VSS/L. The PMC operation under dark/light periods was performed in a 500 mL sequencing batch reactor (SBR) with 8 h cycles, where in the first 4 h of the cycle
Photosynthetic mixed culture operation under dark/light cycles
In this study, a PMC consisting of bacteria and algae that had been enriched under constant illumination was subjected to alternating dark/light conditions in a FF regime, with the culture being fed at the beginning of the dark phase. Fig. 1 shows the evolution of the culture’s performance during an adaptation period of 16 days to the transient illumination conditions. It can be observed that on the 1st day, the PMC was capable of utilizing carbohydrates during the dark phase, likely using it as
Conclusion
A PHA accumulating PMC was enriched in a system with dark/light periods, where a PHA content of 30% was obtained. Moreover, a higher PHB/C yield of 0.90 Cmol/Cmol and twice the specific PHA production rate were achieved in relation to previous works with constant illumination. A direct relationship between light availability and the specific substrate uptake rate was observed, enabling future manipulation of the biomass concentration and illumination intensity in order to further increase PHA
Acknowledgements
The authors would like to acknowledge the financial support from the Fundação para a Ciência e Tecnologia for the Grant SFRH/BD/42085/2007 and Project PEstC/EQB/LA0006/2011.
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