Transient concentrations of NaCl affect the PHA accumulation in mixed microbial culture
Graphical abstract
Introduction
Organic matter recovery from wastewater intends to comply with new regulations from the European Union (Directive 2008/98/EC) which aim at considering wastes as resources to obtain value added products, such as polyhydroxyalkanoates (PHAs).
PHAs are polymers of biological origin, well-known due to their utility for several applications such as raw materials for the production of packaging, medical or building materials. Their advantages rely on being easily biodegradable and the fact that they can be produced from renewable sources [1] or even applied as biofuels [2].
Up to date, most of the research works on PHA have been performed with pure cultures or genetically modified organisms and using valuable industrial by-products as substrate. However, at the moment, the interest is moving to the application of microbial mixed cultures (MMCs), using waste streams as substrate characterized by their large organic matter content. In this latter case, PHA can be produced by MMCs selected by the exploitation of the ecological role of PHA as a microbial storage material [3], when dynamic operational conditions are applied to the system. A selective pressure is imposed, favouring the selection and growth of different microbial cultures able to have clusters of internal carbon as reservoirs [4]. The enhanced capacity of the microbial communities to store PHA under these transitory conditions was confirmed by several authors using as carbon source synthetic substrates or different types of wastewater [5], [6], [7], [8], [9], [10], [11].
When waste streams are used as substrate for PHA production, certain aspects have to be taken into consideration like the presence of salts, the possible existence of inhibitory compounds, seasonal composition variability, etc. Wastewater streams produced in different food industries are suitable as organic matter source for PHA production [12], although in some cases they may contain significant amounts of NaCl or even transient concentrations of this salt [13]. This is the case of the fish-canning wastewaters, which are characterized by a suitable composition for PHA production in terms of organic matter content. This type of wastewater does not have variations just in terms of organic matter content but also with respect to the NaCl concentration, due to the seasonality of the product, the location of the plant, the type of seafood processed and/or the processing procedure. For example, on a seaside factory, the octopus boiling wastewater contains 1.33 g NaCl/g COD while the mussel boiling wastewater has 2.24 g NaCl/g COD, and the fish flour line wastewater has 0.23 g NaCl/g COD [14]. All these products can be processed in the same plant at different moments of the year.
The transitory presence of sodium chloride affects the activity of many different microbial communities in biological wastewater treatment systems [15], [16]. In this sense, the evaluation of the effects of large concentrations of salts has already been the aim of several research works performed using pure cultures. In the specific case of sodium chloride effects, contradictory results have been reported. For example, a progressive reduction of the accumulated PHB, from glycerol as carbon source, by Paracoccus denitrificans and Cupriavidus necator (strain JMP 134) was observed at concentrations above 5 g NaCl/L, reaching an inhibition percentage of 80% at 20 g NaCl/L [17]. On the other hand, Passanha et al. identified a positive effect of the addition of sodium chloride on the PHA storage of C. necator when using acetic acid as substrate. They found that the maximum PHA content was achieved at 9 g NaCl/L while biomass was inhibited at above 15 g NaCl/L. This seems to indicate that the sensitivity to NaCl depends on the type of microorganism involved or on the carbon source used as substrate [18]. Regarding the effect of sodium ion on PHA accumulating organisms, Mozumder et al. observed a detrimental effect over the PHA production of C. necator. PHB production was totally stopped at 10.5 g Na+/L and no biomass growth was observed at 8.9 g Na+/L [19]. Available information shows inconclusive results for the consequences of the presence of NaCl on pure cultures while, to the best knowledge of the authors, these effects have not yet been evaluated on mixed cultures.
For these reasons the main objective of the present study was to research the influence of transient concentrations of sodium chloride, up to 20 g NaCl/L, over the PHA accumulation capacity of a mixed culture. This enriched MMC was previously fed with a mixture of volatile fatty acids (VFAs) containing NaOH for their neutralization, which means a basal concentration of sodium of 0.8 g Na+/L. Estimated kinetic and stoichiometric parameters from obtained PHA-accumulation experiments were used for the evaluation of the culture response.
Section snippets
Experimental set-up and operational conditions
Two bench-scale reactors were used. The first one was used for the selection of the MMC (Enrichment reactor) and the second one for the maximization of biopolymer inside the cells (Accumulation Reactor).
Enrichment of the microbial mixed community
The SBR for the enrichment of the mixed culture was operated during 171 days. Steady state conditions were obtained in the reactor after 18 days from starting up. The average value of the length of the feast phase was of 111.09 ± 27.9 min, corresponding to around 15.4% of the total cycle length (Fig. 1). Previous studies showed that feast/famine ratios shorter than 20% of the cycle length are required for the selection of a MMC with high PHA-accumulating capacity [24], [25].
Ammonia was consumed
Conclusions
The main conclusion obtained from this study is that the presence of added sodium chloride in the feeding media affects the PHA accumulation performance of a MMC non-adapted to saline conditions. These effects are summarized as:
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The amount of stored biopolymer inside the cells decreased when the NaCl concentration increased.
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The HB:HV ratio increased as the concentration of inhibitor also increased. This parameter is a rough indicator of the properties of the produced biopolymer, demonstrating
Acknowledgements
The authors T. Palmeiro-Sánchez, A. Fra-Vázquez, N. Rey-Martínez and A. Mosquera-Corral belong to the Galician Competitive Research Group GRC2013/032, Xunta de Galicia, programme co-funded by FEDER. This work was funded by the Spanish Government through the Plasticwater project (CTQ2011-22675). The authors want to thank M. Orge and M. Dosil for their help with the analytical part of this study.
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