Integrating Hydrothermal Pre-treatment and Vacuum Fermentation for Volatile Fatty Acids and Methane Recovery from Municipal Sludge

Introduction Nowadays, there is an increase interest in recovering value-added products, such as volatile fatty acids (VFA), nutrients, and biomethane from the wastewater. Anaerobic digestion (AD) is a promising technology to reclaim energy from organic matter. Generally, methane produced from the last phase of AD is the main beneficial product. However, by excluding the methane-forming stage, valuable organic acids such as VFAs can be produced by anaerobic fermentation (hydrolysis and acidogenesis). However, relatively long hydraulic retention time (HRT) and low VFA yield are the main challenges associated with fermentation technology. pre-treatment technology with fermentation can be one of the solutions. Hydrothermal pre-treatment (HTP) has attracted industry attention as it accelerates the hydrolysis step, removes pathogens, reduces the viscosity of the sludge, facilitates smoother operation, improves dewatering, and improves the quality of biosolids in terms of odor (Taboada-Santos et al., 2019). Also, to increase organic loading rates without compromising biogas production and organics destruction, recuperative thickening, which involves solid separation of the digestate and solids recirculation to the digester, has been used to increase reactor capacity (Cobbledick et al., 2016a, 2016b; Li et al., 2020). On the other hand, other techniques such as vacuum evaporation is used for sludge dewatering (Yan et al., 2009) and it has been also employed for ammonia and volatile recovery (Nguyen et al., 2011) and hydrogen recovery from yeast fermentation broth (Sonnleitner et al., 2012). Applying a vacuum to the fermenter and or digester allows the decoupling of HRT from solid retention time (SRT) and can facilitate the separation and recovery of resources such as ammonia and VFA, while simultaneously enhancing sludge thickening. However, this technology is limited by low VFA production yield and rate. Therefore, the integration of HTP and vacuum fermentation is proposed and investigated. Integration of the HTP and vacuum fermentation is expected to enhance the degree of solubilization and increase the solids reduction. Furthermore, the effects of vacuum on the interaction of hydrolytic bacteria, fermentative bacteria, and acetogenic bacteria are largely unknown and therefore this study evaluates the microbial community changes in a vacuum and conventional fermentation systems and the integration of the HTP system. Materials and Methods Mixed sludge and anaerobically digested sludge from Ashbridge's wastewater plant were used to run four semi-continuous systems. Fours systems shown in Figure 1 include System (1) conventional fermentation fed with raw TWAS and PS; System (2) Vacuum fermentation fed with raw sludge; System (3) Conventional fermentation fed by pretreated TWAS + raw PS; System (4) Novel vacuum fermentation fed by pretreated TWAS + raw PS. Vacuumed high-grade VFAs and fermentate produced from these systems were further tested for biological nutrient removal (BNR) and AD, respectively. The optimum HTP condition obtained from our previous studies was used to pre-treat TWAS only (temperature: 170 °C, holding time: 30 min, pressure 6 bar) (Kakar et al., 2019). Fermentation tests were set up under thermophilic conditions, in a 3 L reactor. Conventional fermenters operated with HRT=SRT=3 days and vacuum-fermentation with SRT of 3 days and HRT of 1.5 days. Genomic DNA was extracted from the biomass using the PowerSoil DNA isolation kit (MoBio Laboratories Inc.). The DNA samples were sent to the Genome Quebec Research and Testing Laboratory in Montréal, Québec for 16S rRNA gene sequencing (Illumina MiSeq). The DNA samples were amplified using PCR for amplicon preparations. The GenPipes version 4.0.0 (Bourgey et al., 2019) amplicon-seq pipeline was used to perform microbial community analyses. All the water quality analysis were measured according to the standard methods. Results Results revealed that hydrothermal pre-treatment integration with vacuum fermentation significantly improves the overall sludge hydrolysis (p>0.005), while its impact on fermentation was not significant. Figure 2 reports the average COD solubilization for all four systems. The figure shows that both vacuum fermentation systems, either fed by raw or pretreated sludge, had almost similar COD solubilization of 29% and 31% solubilization due to fermentation only. On the other hand, vacuum fermentation demonstrated about 30% improvement in COD solubilization compared to the conventional fermentation (24% vs 31%). As shown in Figure 3, pretreated feed demonstrates higher VFA yield for both cases (vacuum and conventional fermenters). Furthermore, feeding raw sludge exhibited superior results for vacuum fermenter compared to the conventional fermenter (See Figure 3). The average VFA yields in conventional reactors for raw and pretreated feed were 0.12 and 0.21 g COD/g VSS added, respectively, accounting for about 47% improvement due to HTP. The application of the vacuum led to a much higher VFA yield in fermentate compared to condensate. The average VFA yield in the steady-state phase for fermentate, condensate, and overall (fermentate + condensate) were 0.22, 0.011, and 0.23 g COD/g VSS added, respectively. Whereas, in the conventional reactor, the average VFA produced (0.125 g COD/g VSS added) was about 45% lower than the overall (fermentate + condensate) in vacuum fermenter. The microbial community results showed that the most abundant type of bacteria was Coprothermobacteraeota, followed by Synergistetes, Thermotogae, and Firmicutes which are mainly anaerobic bacteria growing in thermophilic conditions (55 „ƒ- 70 „ƒ). The vacuum application showed a great impact on the alpha diversity while for all the sample points the ASV for the vacuum reactors was lower than the conventional reactor by 15-20% (See Figure 4). Considering the steady-state data, the average ASV value for the vacuum and conventional reactors of 14 and 16, respectively, it was evidnced that the hydrothermal pre-treatment impacted the diversity of the microbial communities significantly. The dominant type of bacteria found for pretreated samples from both conventional and vacuum fermenters and raw for the vacuum system were Coprothermobacteraeota and Synergistetes, while on the other hand, Thermotogae and Synergestetes were the most abundant phyla for raw-conventional samples. The effluents of four different fermentation systems were tested to be used as a potential carbon source for denitrification. According to specific denitrification rate (SDNR) results using four effluents, the nitrite accumulation spike was lowest with the HTP vacuum condensate treatment, which indicates that the nitrite reduction (denitrification) started early in the bioreactor fed with HTP vacuum condensate. This is consistent with the above-mentioned finding where overall solubilized COD accumulation was highest in the HTP vacuum bioreactor. Nitrite accumulation was relatively fast with the raw vacuum fermentate (which indicates possibly nitrate removal rate was fast in this reactor). Furthermore, results revealed that condensate of the vacuum fermenters with and without pretreatment has the highest SDNR of 7.6 and 7.2 mg NO3-N/g VSS.h, compared to all other samples, and control (acetate, 6.8 mg NO3-N/g VSS.h), respectively. The results from the biochemical methane potential (BMP) tests of the fermentates that were produced from the four fermenters revealed that the integrated HTP and vacuum fermentation promotes hydrolysis and considerably enhance the methane production yield. The results showed that raw conventional fermented samples and raw vacuum fermented exhibited 28% and 36% increases in methane yield compared to raw (non-fermented sample). Additionally, integrating the HTP and vacuum fermentation increased the methane yields by 53% compared to raw (non-fermented sample). Conclusions Integration of vacuum in the fermentation reactor led to increasing the VFA yields by about 40% compared to the conventional fermentation, and it controlled the acid accumulation due to the continuous extraction of VFA from the system. While the integration of HTP with vacuum fermentation led to reducing the required HRT by 50% (from 3 days to 1.5 days), increasing the VFA yields to 330 mg COD/g VSS compared to 210 mg COD/g VSS for conventional (HTP) reactor, a higher degree of solubilization (45%), and enrichment of the thermophilic microbial communities such as Coprothermobacteraeota and Synergistetes phyla.