Producing Biopolymers from Brewery Wastewater – Enhancing the Yield by Addition of Calcium Carbonate

Producing Biopolymers from Brewery Wastewater – Enhancing the Yield by Addition of Calcium Carbonate C. M. Laumeyer* and H. Steinmetz* * Department of Resource Efficient Wastewater Technology, RPTU University of Kaiserslautern-Landau, PaulEhrlich-Str. 14, 67663 Kaiserslautern, Germany (E-mail: [email protected]; heidru[email protected]) Abstract The brewing industry generates significant volumes of nutrient-rich wastewater, presenting both environmental challenges and untapped opportunities. With the aim to valorize this waste stream, the biodegradable biopolymer polyhydroxyalkanoate (PHA) was produced in a three-step process, exploring a process improvement by the addition of calcium to increase the overall yield, based on findings from Estévez-Alonso et al. (2022) who used a synthetic feedstock. After producing a volatile fatty acid rich feedstock from brewery wastewater, waste activated sludge (WAS) from the brewery’s wastewater treatment plant was supplied with this feedstock in a feast-famine regime to produce a mixed microbial culture (MMC) rich in PHA-producing organisms. The enrichment stage was continuously operated as triplicate lab-scale reactors of 10 L volume, supplying the VFA-rich feedstock every 6h. Additionally, 1 g CaCO3 was supplied to the enrichment reactors on weekdays. To assess the PHA productivity, 24h-long batch accumulations were performed, supplying the feedstock by pulse-feeding with 100 mg CODVFA L-1Feed-1. Results showed, that the PHA content of the accumulations on day 7 were comparable with an average of 35.8±9.9 gPHA/gVSS in the reactors supplied with CaCO3 (WAS+ CaCO3) compared to 36.57 gPHA/gVSS in the WAS-reactors. However, the concentration of PHA in the WAS+CaCO3 accumulation reactors was clearly higher (153±30 mg L-1) compared to the WAS-reactor (87 mg L-1) caused by a higher VSS concentration. The results show that the addition of CaCO3 has a positive impact on the overall production of PHA from waste streams. Future experiments will explore the optimal amount of CaCO3. Keywords Carbon recovery; renewable resources; VFA; industrial wastewater; polyhydroxyalkanoates; PHA composition INTRODUCTION With an increase of the society’s awareness to environmental problems, there has been a rise within the field of science regarding biotechnological solutions. In this realm, the interest in biodegradable alternatives to conventional plastics has accelerated the research on biopolymers. One promising biopolymer is polyhydroxyalkanoate (PHA), which serves as an energy storage for several different microorganisms. While industrial PHA-production uses pure bacterial cultures and purified carbon sources, there is a push to utilize waste streams and mixed microbial cultures (MMC) making the process more robust and eliminating the problem of competing for resources (Reis et al. 2003; Koller et al. 2017). Due to fluctuations in the composition and availability of different waste streams that influence the final product, the complexity of the process operation increases (Morgan-Sagastume et al. 2020; Khatami et al. 2021). Furthermore, process control does not only focus on the PHA composition but also on the yield that is achieved by using different waste streams. While the enrichment is successful regarding the PHA producing bacteria, process controls to enhance the overall yield are still being explored. Estévez-Alonso et al. (2022) discussed the benefits of adding calcium during the PHA production to enhance the selective growth in the MMC using acetate as a feedstock. In their study the addition of Ca(OH)2 to the acetate feedstock resulted in higher PHA yields compared to NaOH, KOH and Mg(OH)2. However, they state that this approach is not feasible when using waste streams, as they may already contain calcium (Estévez-Alonso et al. 2022). Using a brewery wastewater derived feedstock, the conducted experiments aim to assess the feasibility of adding calcium carbonate (CaCO3) during the enrichment of a MMC to increase the PHA yield and compare it to an enrichment without CaCO3. MATERIALS AND METHODS To produce biopolymers from brewery wastewater, in a first step wastewater from a local brewery was mixed with 10% inoculum by volume from an upflow anaerobic sludge blanket reactor of the brewery’s wastewater treatment plant (WWTP) and anaerobically acidified for 6 days at 35°C. After 6 days a solid-liquid separation was performed to separate the supernatant rich in volatile fatty acids (VFAs). Ionchromatography allowed an analysis of the different VFAs (acetic- (HAc), propionic- (HPr), iso-butyric- (HIBu), butyric- (HBu), iso-valeric- (HIVa), valeric- (HVa) and caproic (HCa) acid) in the so obtained substrate for PHA production. After an initial analysis of the VFA composition, the substrate was spiked with commercial VFAs of analytical standard, keeping the initial VFA composition profile to facilitate several PHA production experiments using the same feedstock and allow comparability. The substrate’s pH was increased to 7 using NaOH (32%) to prevent a pH shock of the biocenosis. Previous analysis of the brewery derived substrate showed a nutrient deficiency, which is why NH4Cl and KH2PO4 were added to the feedstock for the enrichment in a ratio of 100:5:1 (CODVFA:NH4-N:PO4-P). Additionally, 2 mL of a micronutrient solution consisting of 1.5 g FeCl3·6H2O, 0.15 g H3BO3, 0.03 g CuSO4·5H2O, 0.018 g KI, 0.12 g MnC12·4H2O, 0.06 g Na2MoO4·2H2O, 0.12 g ZnSO4·7H2O, 0.15 g CoC12·6H2O and 10 g EDTA (Smolders et al. 1994) were added per litre substrate. The actual PHA production was performed in a two-step process consisting of a continuous operated enrichment stage and targeted 24h-long batch accumulations in triplicates at laboratory scale. The 10 L enrichment reactors were inoculated with excess sludge from the brewery’s WWTP. The produced VFA-rich feedstock was supplied in a feast-famine-regime with an organic loading rate (OLR) of 1 g L-1d-1 at aerobic conditions and a 6h cycle. Additionally, 1 g CaCO3 was added on week days (Monday-Friday) to each enrichment reactor. Overhead stirrers allowed additional mixing of the reactor. Every 6h 1.25 L reactor volume was withdrawn, before a fresh feed of substrate was supplied. Every second cycle a 45 min. sedimentation was implemented to prevent a loss of solids. For the batch accumulations, the feedstock was supplied as pulse-feeds supplying 100 mg CODVFAL-1 per feed. The first accumulation using the excess sludge allowed a measurement of the initial PHA accumulation potential. For the batch accumulations on day 7 and day 14 the fully mixed withdrawal of the enrichment reactors was used. Due to analytical sampling to measure PHA, VFA, DOC, TSS and VSS as well as nutrients in form of TNb, NH4-N and PO4-P, the accumulations started with 0.5 L. After centrifugation and filtration through 0.45 µm regenerated cellulose filters, photometric Hach cuvette test kits were used to analyse the nutrient concentration (NH4-N, PO4-P) while DOC and TNb were analysed using Elementar Vario TOC cube. Total suspended solids (TSS) and volatile suspended solids (VSS) were measured according to DIN 38 409H 2–2 using black ribbon filters (589/1, Whatman®) in the beginning and the end of the accumulation. The analysis of the PHA content and composition was measured as described in Laumeyer et al. (2025) using gas chromatography with downstream flame ionisation detector (GCFID, Agilent 8860 design; Agilent Technologies™). RESULTS AND DISCUSSION Composition of the produced feedstock The spiked feedstock used in the experiment had a concentration of 2.17 g CODVFA/L and the VFAs represented 89% of the dissolved organic carbon (DOC). The composition of the feedstock is depicted in Table 1. The feedstock consisted mainly of HPr with 30%, 27% HIVa and 23% HIBu and minor amounts of HCa (8%), HAc (5%), HBu (5%) and HVal (3%). As the ratio of VFAs with an even number of carbon atoms vs. odd having an influence of the PHA composition (Lemos et al. 2006; Albuquerque et al. 2007), this feedstock showed a predisposition to be converted to PHV, as 59% of the carbon was provided in form of oddly numbered VFAs (HPr, HVa, HIVa), while 41% of the feedstocks’ carbon was provided in the form of even numbered VFAs. Table 1. Composition of feedstock produced from brewery wastewater by anaerobic acidogenic fermentation used to produce the biopolymer PHA. Parameter Unit Brewery derived feedstock CODVFA g/L 2.17 DOC g/L 0.73 C-VFA g/L 0.65 C-VFA/DOC % 89 TNb (total N bound) mg/L 107 NH4-N mg/L 100.2 PO4-P mg/L 22.5 CODVFA:NH4-N:PO4-P [-] 100:4.6:1.0 Acetic acid (HAc) g/L 0.041 Propionic acid (HPr) g/L 0.291 Iso-butyric acid (HIBu) g/L 0.270 Butyric acid (HBu) g/L 0.060 Iso-valeric acid (HIVa) g/L 0.365 Valeric acid (HVa) g/L 0.036 Caproic acid (HCa) g/L 0.121 CODVFA: Concentration of volatile fatty acids (VFA) converted to chemical oxygen demand. C-VFA: Concentration of carbon in form of VFA. Development of the PHA content and composition While the composition of the brewery derived feedstock consisted mainly of VFAs with an odd number of C-atoms (59%) which tend to be converted to PHV (Lemos et al. 2006; Albuquerque et al. 2007), the results of the monomeric composition show mainly HB-monomers throughout the performed accumulations, as depicted in Figure 1. Figure 1. Development of the PHA composition of the produced PHA regarding its monomer composition in the triplicates. The molar content is divided into the monomers hydroxybutyrate (HB), hydroxyvalerate (HV) and hydroxy hexanoate (HH), with standard deviation of ± 1% for HB & HV. Effect of the addition of CaCO3 to the enrichment The results of the enrichment operated with addition of CaCO3 is depicted in comparison with the results obtained without CaCO3 in Figure 2. While Figure 2 A shows the percentage of PHA regarding the VSS concentration, Figure 2 B shows the concentration of PHA in mg L-1. What is striking, is that the even though the percentage of PHA seems to be quite similar in both enrichment set-ups, the resulting PHA concentration is increased on day 7 by 75% and on day 14 by 27% in the accumulations that were performed using the CaCO3-enriched biomass caused by a higher VSS-concentration. This is in line with the results reported by Estévez-Alonso et al. (2022), who state the addition of calcium leads to an increase of both the PHA production rate and higher biomass production, caused by lower cellular energy requirements. 0% 25% 50% 75% 100% 0 7 14 Molar content Enrichment Time [d] HB HV HH Figure 2. Development of the PHA content of the produced PHA in batch accumulations on day 0, 7 and 14 using either waste activated sludge with the addition of 1 g CaCO3 (WAS+CaCO3) or without (WAS). Figure 2 A shows the percentage of PHA within the volatile suspended solids (VSS) and Figure 2 B shows the results calculated to the concentration of PHA in mg/L. Overall, the results show a promising improvement of the PHA production yield by the addition of CaCO3 when using acidified brewery wastewater as a feedstock. The mechanisms and the influence of the CaCO3 on the composition of the produced PHA need to be investigated further. 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