Elsevier

New Biotechnology

Volume 56, 25 May 2020, Pages 140-148
New Biotechnology

Full length Article
High rate selection of PHA accumulating mixed cultures in sequencing batch reactors with uncoupled carbon and nitrogen feeding

https://doi.org/10.1016/j.nbt.2020.01.006Get rights and content

Highlights

  • Uncoupled carbon and nitrogen feeding enhances the biomass PHA storage response.

  • The OLR has a strong impact on storage response and PHA volumetric productivity.

  • The MMC selected with uncoupled feeding reached more than 70 % g PHA/g VSS.

  • High OLR and uncoupled C-N feeding make the accumulation stage unnecessary.

  • Polymer composition is strongly affected by the OLR applied.

Abstract

The selection and enrichment of a mixed microbial culture (MMC) for polyhydroxyalkanoates (PHA) production is a well-known technology, typically carried out in sequencing batch reactors (SBR) operated under a feast-famine regime. With a nitrogen-deficient carbon source to be used as feedstock for PHA synthesis, a nutrient supply in the SBR is required for efficient microbial growth. In this study, an uncoupled carbon (C) and nitrogen (N) feeding strategy was adopted by dosing the C-source at the beginning of the feast and the N-source at the beginning of the famine, at a fixed C/N ratio of 33.4 g COD/g N and 12 h cycle length. The applied organic loading rate (OLR) was increased from 4.25 to 8.5 and finally to 12.725 g COD/L d. A more efficient selective pressure was maintained at lower and intermediate OLR, where the feast phase length was shorter (around 20 % of the whole cycle length). However, at the higher OLR investigated, the PHA content in the biomass reached a value of 0.53 g PHA/g VSS at the end of the feast phase, as a consequence of the increased C-source loaded per cycle. Moreover, 2nd stage PHA productivity was 2.4 g PHA/L d, 1.5 and 3.0-fold higher than those obtained at lower OLR. The results highlight the possibility of simplifying the process by withdrawing the biomass at the end of the feast phase directly to downstream processing, without a need for the intermediate accumulation step.

Introduction

In recent years, the production of fully biodegradable biopolymers from renewable resources has become widespread [[1], [2], [3]]. Polyhydroxyalkanoates (PHA) are polyesters of hydroxyalkanoic acids, naturally produced as storage carbon (C) sources by different species of PHA-producing microorganisms [4]. They are completely biodegradable and can be produced from renewable resources and waste material, showing elastomeric and thermoplastic properties comparable with traditional plastics [5]. Mixed microbial cultures (MMCs) have been proposed as a cost-effective means of producing PHA from renewable resources (i.e. activated sludge and organic wastes) through the selection of PHA-storing microorganisms, obtained applying alternate dynamic feeding conditions [[6], [7], [8]]. High selective pressure for the PHA-storing bacteria in activated sludge has been obtained by setting periodic alternating feast (C feeding) and famine (absence of C sources) conditions [[9], [10], [11], [12], [13]]. Establishing these conditions enables a physiological adaptation of microbial species, leading to the selection of microorganisms which produce and accumulate PHA as intracellular C source.

As extensively reported previously [14,15], the typical parameter for a good selection of PHA-storing biomass is the ratio between the length of the feast phase and the whole cycle length, which should be lower than 20 %. A sequencing batch reactor (SBR) is generally used for the selection of the PHA-accumulating biomass, as it is possible to apply the required dynamic feeding strategy [16]. The step following is the PHA production, usually conducted in an accumulation batch reactor inoculated with the selected PHA-producing biomass. The use of renewable and fermentable feedstock can lead to a significant reduction of costs in the overall process [[17], [18], [19], [20]]. Hence, VFA-rich streams are typically used as feedstock for MMC-PHA accumulation processes. These streams may contain varying levels of nutrients (N and P) that can affect process performance. Nutrient deficient waste streams as feedstock for both the selection and accumulation steps may be used even though nutrient limitations can cause an unstable growth of PHA-producing microorganisms in the SBR [[21], [22], [23], [24]]. On the other hand, it has been demonstrated that N limitation during the accumulation step can substantially increase the production performances in terms of PHA storage yield and PHA final content [12,[21], [22], [23], [24], [25]].

A focus of current research has been the optimization of N supply [17]. A particular application of the uncoupled feeding strategy has been reported to show how the latter allows using specific feedstock (e.g. 1,3-propanediol), which was found to be unsuitable for PHA production from MMC under coupled C and N feeding conditions [26] whereas, in previous studies [27,28], N and C feeding have been uncoupled, stimulating a PHA storage response during the feast phase (in the absence of N) and microbial growth in the famine phase (adding nitrogen). The impact of regulation of N feeding on the process has been evaluated firstly in a laboratory-scale SBR with a cycle length of 6 h under an applied organic load rate (OLR) equal to 8.5 g COD/L d (Chemical Oxygen Demand, COD) [27]. Two SBR runs were performed with the C source fed at the beginning of the SBR cycle simultaneously with the N source (coupled feeding strategy) or with the latter fed at the end of the feast phase (uncoupled feeding strategy). As a main result, it was found that PHA content at the end of the feast phase was doubled with uncoupled feeding. Accordingly, in the present study the effect of the applied OLR has been investigated maintaining the uncoupled C and N feeding strategy, with a fixed C/N ratio. The SBR was operated with a 12 h cycle length and at three OLRs (4.25, 8.5 and 12.75 g COD/L d), employing a synthetic mixture of acetic and propionic acids as C feeding solution. The OLR was explored up to 12.75 g COD/L d, a level rarely considered in the literature, and was varied in order to evaluate its effect on both PHA storage properties and process productivity.

Section snippets

Sequencing batch reactor for MMC selection/enrichment

The selection and enrichment of PHA-accumulating biomass was performed in a fully aerobic SBR (1.0 L working volume), inoculated with an activated sludge from “Roma Nord” full-scale wastewater treatment plant (Rome, Italy). A mechanical impeller was used for mixing of the culture medium with O2 provided through air pumps connected to ceramic diffusors. The operating cycle length was set at 12 h, in all three SBR runs. The cycle structure was composed follows: initial phase of carbon (C) source

Selection and enrichment of the mixed microbial culture in SBR: the effect of applied OLR

Three runs were carried out following the uncoupled C- and N-sources feeding strategy in order to maximize the storage yield in the feast phase and optimize the selective pressure on the culture. Therefore, the N-source was fed at the beginning of the famine (after complete VFA depletion) and the ammonia was used for the growth of PHA-storing organisms. The trend of the main parameters during a typical SBR cycle was reported in Fig. 1, Fig. 2, Fig. 3 for the three OLRs adopted. In both Figs. 1a

Conclusions

This work has highlighted the possibility of significantly enhancing PHA production in an SBR operating with uncoupled C and N feeding by determining the optimal operating conditions. The OLR applied has been shown to have a significant impact on the aerobic MMC selection/enrichment, and, in consequence, on storage performances and productivity. An ideal selective pressure was maintained at up to 8.5 g COD/L d, and partially reduced at higher OLR. The relatively high PHA content achieved at the

Acknowledgements

This work was financially supported by the SMART-PLANT (GA 690323) project in the European Union Horizon 2020 program.

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