Biopolymer production potential from pickle brine effluent through microbial processes

Abstract

The use of plastics is increasing every year around the world. This increase brings along both the waste problem and the raw material problem. Due to the waste problem, political and environmental problems are experienced worldwide. Since conventional plastics cannot be degraded in nature, their disposal is being more challenging and costly. When they are discarded, they can take hundreds of years to decompose, leading to a buildup of plastic waste in landfills and oceans. This plastic waste poses a threat to wildlife and the environment. In addition, the raw material for conventional plastics in which petroleum derivatives are non-renewable sources and they will eventually be depleted one day. In this context, use of alternative sources is critical for sustainable development. It is especially important that these resources, which can be offered as alternatives, should be biodegraded in nature and renewable sources. Biopolymers have been considered as new generation bioplastics however studies for optimum cost with high efficiency is still required. In this thesis, the production of polyhydroxyalkanoate (PHA) biopolymers, which are categorized as both biodegradable and bio-based, with pickle brine effluent was studied. PHA is a type of natural biopolymer that is produced by microorganisms and is known for being eco-friendly. PHAs have several advantages over conventional plastics with their characteristics of biodegradibility, renewable, biocompatibility and versatility. Metabolic production of PHA can be done by both pure culture microorganisms and mixed culture microorganisms. Pure culture microorganisms necessitate specific substrates, sterile environments, and cause high cost. There is a high risk of contamination, and the sterilization is required to prevent this contamination. Mixed microbial culture can be a more sustainable and cost-effective approach compared to pure culture methods. Mixed microbial cultures are less prone to contamination and more suitable for large-scale production. They have a higher diversity of microorganisms, which can lead to a wider range of PHA types and properties. And they can utilize low-cost carbon sources, such as wastewater and organic waste, which can reduce the overall production costs and contribute to circular economy. In this thesis, the production of PHA from pickle industry wastewater is studied with mixed microbial culture. In PHA production with mixed microbial culture (MMC), the carbon feedstock is fermented to produce a mixture of soluble fermentation products (SFP). Then, a community of PHA-storing bacteria is selected and enriched with various enrichment methods. Lastly, the selected culture is utilized to produce PHA, whereby SFP is added to the culture under conditions that limit growth to ensure the culture reaches its highest PHA capacity. In this thesis, the pickle brine effluent was used as feedstock for PHA production. In this context food waste is used for biopolymer production in which waste will be converted to high-value added product. This approach will both minimize the use of non-renewable resources and contribute to circular economy. The featuring characteristics of the pickle brine effluent is that it contains high organic acid content and high salinity. In order to ease the acclimation of the mixed microbial culture to saline conditions, the inoculum was obtained from the pickle industry wastewater treatment plant. The mixed microbial culture (MMC), was first acclimated to high salinity and further enriched for PHA producing MMC with aerobic dynamic feeding (ADF) regime. High salinity of the pickle brine effluent is used for the stress condition for ADF regime. Since the fermented pickle brine effluent includes various organic acids with high concentration, fermentation step was not required for PHA production. The pickle brine effluent had high organic and chloride content, with an average of 19,050±700 mg/L chemical oxygen demand (COD) and 39,175±1,155 mg/L chloride (Cl-), respectively. Total kjeldahl nitrogen (TKN), total phosphorus (TP) concentrations and pH values were 445 ± 18 mg N/L, 32± 46 mg P/L and 2.95±0.15, respectively. The organic acid content of the pickle brine effluent was 1,035±290 mg/L of acetic acid, 8,365±300 mg/L of lactic acid, 5,680±550 mg/L of propionic acid and 490±155 mg/L of succinic acid. In this study, for the experimental set-up, working volume of the the sequencing batch reactor (SBR) was selected as 2 L. Initially, SBR was operated as 1 cycle/day for the acclimation of the MMC to high salinity. The total suspended solids (TSS) concentration and volatile suspended solids (VSS) concentration of the inoculum was 8,065±605 mg/L, and 5,775±240 mg/L, respectively. After the inoculum was adapted to the pickle brine effluent, the culture enrichment process was started. Aerobic dynamic feeding (feast/famine regime) was used for the enrichment. SBR was fed with pickle brine effluent with 3,800 mg COD/L and 7,850 mg Cl-/L per cycle. The organic loading rate (OLR) of the reactor was 7.6 g COD/L.day. Also the pH of the feeding solution was adjusted to 6 to provide optimum environmental conditions and the SBR was kept in an isothermal room at 22°C. One cycle duration was 720 minutes (12 hours) consisted of 7 minutes of feeding, 630 minutes of aeration, 80 minutes of settling and 10 minutes of discharge. Waste sludge was discharged once per day as 500 mL to sustain a sludge retention time of 4 days. The ratio of initial volume to the total volume was 0.5. The SBR was operated for 209 days. The performance of the system was monitored by taking samples weekly from the SBR. Within the scope of SBR performance monitoring studies, volatile suspended solids, organic acid and PHA measurements were done in regular terms. The average volatile solids concentrations in the SBR were measured as 5,200±1,175 mg MLVSS/L at steady state conditions. In order to see the maximum PHA storage potential of the system, in-cycle monitoring studies were carried out. Thus, the SBR was monitored during a whole period of a cyle time (12 hours). The PHA content of the biomass was were found to be 0.48 mg COD/mg VSS, 0.52 mg COD/mg VSS and 0.59 in mg COD/mg VSS, respectively. These results are comparable with the literature. According to in-cycle analysis, HB and HV monomers of PHA biopolymer were found to be 98% (w/w) and 2% (w/w), respectively, and HB was observed as the major monomer. The polymer composition depends on the substrate composition, and it is known that HB is synthesized as a precursor, usually with acetic acid lactic acid-containing substrate. In this study, acetate and lactate were the main organic acid fraction utilized by the enriched culture for PHA production, while the propionic acid was used for growth. In addition, a batch experiment was also carried out to observe the effect of the organic loading rate on PHA storage yield. In this experiment, the organic feeding was 9,500 mg COD/L and the PHA content of the biomass was measured as 0.42 mg COD/mg VSS. The chloride concentration was quite high as 19,600 mg Cl-/L compared to the SBR with 3,800 mg COD/L and 7,850 mg Cl-/L feeding. It was observed that, high salinity reduced the reactor performance with lower PHA productions. Consequently, the results indicate that pickle brine effluent is a promising alternative for PHA biopolymer production with the optimization of the system.