Understanding Delayed PHA Accumulation in Industrial Wastewaters: A Modelling Approach

Understanding Delayed PHA Accumulation in Industrial Wastewaters: A Modelling Approach O. Karahan Ozgun* and S. Yilmaz* * Istanbul Technical University, Faculty of Civil Engineering, Department of Environmental Engineering, 34469, Maslak, Istanbul, Turkey (E-mail: [email protected]; [email protected]) Abstract Polyhydroxyalkanoates (PHA) are promising biopolymers that serve as sustainable alternatives to conventional plastics. Industrial wastewater represents a costeffective and abundant substrate for PHA production, offering dual benefits of resource recovery and waste management. Respirometric experiments conducted with mixed microbial cultures using wastewater from the textile and wheat processing industries revealed a delayed PHA storage response under certain environmental conditions, posing challenges to process optimization. Based on the interpretation of these experimental results, a metabolically structured model has been developed to accurately describe the observed kinetics. This model provides valuable insights into the factors contributing to the delay and offers a predictive framework to enhance PHA production efficiency. By addressing these challenges, the study aims to advance the feasibility of industrial-scale PHA production using diverse wastewater sources. Keywords Delayed storage response; Industrial wastewater; Mathematical modelling; Mixed microbial cultures (MMC); Polyhydroxyalkanoates (PHA); Respirometric assessment INTRODUCTION The production of PHA using MMCs has gained significant attention as a sustainable approach for bioplastic synthesis. Unlike processes utilizing pure carbon sources, where PHA accumulation occurs almost instantaneously, employing industrial wastewater as a substrate introduces additional complexity (Tamang et al., 2021). Industrial wastewater comprises a heterogeneous mixture of organic compounds and volatile fatty acids (VFAs), requiring microorganisms to metabolize these diverse carbon sources through the activation of specific enzymes and metabolic pathways (Mezzolla et al., 2018; Zhang et al., 2015). This complexity is further compounded by the variability and often harsh conditions inherent to wastewater, which impose metabolic stress on microorganisms (Tu et al., 2020). This results in a delay in the initiation of PHA storage as microorganisms undergo an adaptation phase. While VFAs are preferred substrates for PHA synthesis, their consumption necessitates the concurrent activation of both energy metabolism and PHA biosynthetic pathways, which may slow down the overall process (Yu et al., 2002). Given these challenges, understanding the dynamics of PHA storage under such conditions is crucial. The balance between intracellular energy and carbon fluxes, influenced by factors such as the availability of carbon sources, pH, and oxygen levels, governs the overall efficiency of PHA accumulation. However, due to the complex and variable nature of industrial wastewater, predicting and optimizing these processes becomes increasingly difficult. To address this complexity, mathematical modelling plays a crucial role in elucidating the kinetics and uncertainties involved. Robust models can be developed to simulate the dynamics of microbial behavior, identify rate-limiting steps, and optimize the conditions for efficient PHA production. This approach not only aids in understanding the metabolic pathways but also provides insights for the scalable and sustainable production of bioplastics from industrial wastewater. MATERIALS AND METHODS Textile industry wastewater samples were taken from a textile finishing industry located in Tekirdag, Turkey, with an average production capacity of 45 tons per day. The wastewater was supplied from the effluent of the knit fabric 95°C reactive dyeing with bleaching process consisting of 10 batch steps in the production process. Wheat processing industry wastewaters are taken from a bulgur production factory in Türkiye. The facility is located in Mersin, Turkey. An average of 375 tons/day of wastewater is generated in the processes carried out in the wheat processing, namely bulgur producing factory with a capacity of 340 tons.bulgur/day. The characteristics of the wastewater are shown in Table 1. Activated sludge from a municipal wastewater treatment plant in Istanbul was used as the inoculum source for the acclimation reactor and acclimated inoculum was used for starting up the enrichment reactor, which was operated as a 2 L sequencing batch reactor (SBR) with an MLVSS of 3 g VSS/L and 0.4 g COD/L/cycle. Two-liter aerated batch reactors were used to measure the oxygen uptake rate (OUR) of MMC enriched for PHA production. OUR analyses were performed using an Applitek Ra-COMBO 1000 respirometer. The OUR profile in an aerated batch reactor was simulated using AQUASIM® for the solution of the nonlinear mass balance equations involved. Table 1. Wastewater characteristics Parameter Unit Bulgur ww Textile ww pH - 4.28 4.54 TSS mg/L 3160 <10 tCOD mg/L 16825 695 sCOD mg/L 13525 630 TP mg/L 37 <0,1 orto-P mg/L 24 <0.1 TKN mg/L 321 <5 NH3-N mg/L 18 <5 RESULTS AND DISCUSSION It has been observed that the storage response of the acetate fed system was immediate and the end of the storage phase was at the point where the first phase oxygen uptake rate (OUR) response incorporated of PHA storage was completed as seen in Fig. 1. However, when the same enriched mixed microbial culture (MMC) capable of PHA storage was fed with textile wastewater with the addition of acetate, the storage response was delayed and started after the completion of the first phase OUR response (Fig.2). This shows that although the uptake rates of the external substrate and oxygen for the substrate utilization process were similar in the two-given respirometric tests, synthesis of PHAs were delayed in the system fed with textile wastewater, containing mainly acetate, due to the inhibition of PHA synthesis with industrial wastewater. Similar responses were observed for fermented bulgur production effluents. These experimental findings are the first examples of delayed storage response reported in literature for industrial wastewaters, although there are several studies showing inhibitory effects of salt (NaCl) content in the feed source on PHA storage. Figure 1. The respirometric response, COD utilization & PHA accumulation in the acetate fed system Figure 2. The respirometric response, COD utilization & PHA accumulation in the textile wastewater + acetate fed system The modelling studies were conducted in order to simulate the OUR and PHA storage responses of the MMC fed with industrial wastewaters. The metabolic model structure, kinetic and stoichiometric coefficients for delayed storage response were revealed. The results of this modelling study will be useful for setting up strategies for maximum PHA accumulation in the scale -up applications and adjusting the design considerations of biorefineries targeting PHA production from different industrial waste streams. REFERENCES Mezzolla, V., D’Urso, O. F., Poltronieri, P. 2018. Role of PhaC type I and type II enzymes during PHA biosynthesis. Polymers 10(8), 910. Tamang, P., Arndt, C., Bruns-Hellberg, J., Nogueira, R. 2021. Polyhydroxyalkanoates production from industrial wastewaters using a mixed culture enriched with Thauera sp.: Inhibitory effect of the wastewater matrix. 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