Effects of Different pH Control Strategies on Microalgae Cultivation and Nutrient Removal from Anaerobic Digestion Effluent.

This study investigated nutrient removal from anaerobic digestion effluent by cultivating mixed-culture microalgae enriched from anaerobic sludge under different pH conditions: RUC (uncontrolled), R7-8 (maintained at 7-8), and R<8 (maintained below 8). Significant amounts of NH4+-N were lost by volatilization in RUC cultures due to increased pH values (≤8.6) during the early period of cultivation. The pH control strategies significantly affected the biological NH4+-N removal (highest in R7-8), microalgal growth (highest in R7-8), biomass settleability (highest in R<8), and microalgal growth relative to bacteria (highest in R<8) in the cultures. Parachlorella completely dominated the microalgal communities in the inoculum and all of the cultures, and grew well at highly acidic pH (<3) induced by culture acidification with microalgal growth. Microalgae-associated bacterial community structure developed very differently among the cultures. The findings call for more attention to the influence and control of pH changes during cultivation in microalgal treatment of anaerobic digestion effluent.

Designing a marine outfall to reduce microbial risk on a recreational beach: Field experiment and modeling.

A marine outfall can be a wastewater management system that discharges sewage and stormwater into the sea; hence, it is a source of microbial pollution on recreational beaches, including antibiotic resistant genes (ARGs), which lead to an increase in untreatable diseases. In this regard, a marine outfall must be efficiently located to mitigate these risks. This study aimed to 1) investigate the spatiotemporal variability of Escherichia coli (E. coli) and ARGs on a recreational beach and 2) design marine outfalls to reduce microbial risks. For this purpose, E. coli and ARGs with influential environmental variables were intensively monitored on Gwangalli beach, South Korea in this study. Environmental fluid dynamic code (EFDC) was used and calibrated using the monitoring data, and 12 outfall extension scenarios were explored (6 locations at 2 depths). The results revealed that repositioning the marine outfall can significantly reduce the concentrations of E. coli and ARGs on the beach by 46-99%. Offshore extended outfalls at the bottom of the sea reduced concentrations of E. coli and ARGs on the beach more effectively than onshore outfalls at the sea surface. These findings could be helpful in establishing microbial pollution management plans at recreational beaches in the future.

Individual and combined effects of magnetite addition and external voltage application on anaerobic digestion of dairy wastewater.

Direct interspecies electron transfer (DIET) between exoelectrogenic fatty acid oxidizers and electrotrophic methanogens plays an important role in keeping the overall anaerobic digestion (AD) process well-balanced. This study examined the individual and combined effects of two different DIET-promoting strategies, i.e., magnetite addition (20 mM Fe) and external voltage application (0.6 V), in continuous digesters treating dairy wastewater. Although the strategies were both effective in enhancing the process performance and stability, adding magnetite had a much greater stimulatory effect. External voltage contributed little to the methane yield, and the digester with magnetite addition alone achieved stable performance, comparable to that of the digester where both strategies were combined, at short hydraulic retention times (down to 7.5 days). Diverse (putative) electroactive microorganisms were significantly enriched under DIET-promoting conditions, particularly with magnetite addition. The overall results suggest that magnetite addition could effectively enhance AD performance and stability by promoting DIET-based electro-syntrophic microbial interactions.

Temperature Effects on Methanogenesis and Sulfidogenesis during Anaerobic Digestion of Sulfur-Rich Macroalgal Biomass in Sequencing Batch Reactors.

Methanogenesis and sulfidogenesis, the major microbial reduction reactions occurring in the anaerobic digestion (AD) process, compete for common substrates. Therefore, the balance between methanogenic and sulfidogenic activities is important for efficient biogas production. In this study, changes in methanogenic and sulfidogenic performances in response to changes in organic loading rate (OLR) were examined in two digesters treating sulfur-rich macroalgal waste under mesophilic and thermophilic conditions, respectively. Both methanogenesis and sulfidogenesis were largely suppressed under thermophilic relative to mesophilic conditions, regardless of OLR. However, the suppressive effect was even more significant for sulfidogenesis, which may suggest an option for H2S control. The reactor microbial communities developed totally differently according to reactor temperature, with the abundance of both methanogens and sulfate-reducing bacteria being significantly higher under mesophilic conditions. In both reactors, sulfidogenic activity increased with increasing OLR. The findings of this study help to understand how temperature affects sulfidogenesis and methanogenesis during AD.

Co-feeding spent coffee grounds in anaerobic food waste digesters: Effects of co-substrate and stabilization strategy.

Anaerobic digestion of spent coffee grounds (SCG) is considered disadvantageous, particularly under mono-digestion conditions, owing to slow degradation and nutrient imbalance. This study investigated the effect of co-feeding of SCG at a low ratio into food waste (FW) digesters, with the aim to determine whether SCG can be effectively treated and valorized using the spare capacity of existing digesters. Duplicate reactors showed stable performance under FW mono-digestion conditions but manifested severe deterioration in three volume turnovers after co-feeding of SCG (FW:SCG at 10:1 on a volatile solids basis). The reactors failed to recover despite repeated interrupted feeding and stabilization, and Ulva was added (FW:SCG:Ulva at 20:2:1) for nutrient supplementation. The two reactors subjected to different stabilization strategies (i.e., timing and intervals of interrupted feeding) responded differently to Ulva co-feeding: one recovered and maintained stable albeit suboptimal performance, whereas the other failed. Furthermore, the microbial communities developed differently in the reactors.

Nutrient removal and microalgal biomass production from different anaerobic digestion effluents with Chlorella species.

Potential of microalgal cultivation as an alternative approach to the treatment of anaerobic digestion (AD) effluents was examined using two representative Chlorella species, Chlorella vulgaris (CV) and Chlorella protothecoides (CP). Both species effectively removed NH4+-N from the AD effluents from four digesters treating different wastes under different operating conditions. In all experimental cultures on the AD effluents, NH4+-N (initial concentration, 40 mg/L) was completely removed within 10 days without residual NO3--N or NO2--N in batch mode. Compared to CP, CV showed greater biomass and lipid yields (advantageous for biodiesel production), regardless of the media used. Prolonged nitrogen starvation significantly increased the lipid accumulation in all cultures on the AD effluents, and the effect was more pronounced in the CV than in the CP cultures. On the other hand, compared to CV, CP showed significantly faster settling (advantageous for biomass harvesting) in all media. Our results suggest that the Chlorella cultivation on AD effluents under non-sterile, mixed-culture conditions may provide a viable way to manage and valorize the problematic effluents. Diverse bacteria derived from the AD effluents co-existed and presumably interacted with the Chlorella species in the cultures.

Potential of mixed-culture microalgae enriched from aerobic and anaerobic sludges for nutrient removal and biomass production from anaerobic effluents.

This study examines the potential of the mixed-culture microalgal consortia enriched from aerobic sludge (AeS) and anaerobic sludge (AnS) with regard to nutrient removal and biomass production from four different anaerobic digestion (AD) effluents. Both the inocula achieved the complete removal of the NH4+-N (initial concentration of 40 mg/L) within 14 days from all the effluents. The AeS cultures showed faster and greater microalgal growth, although the NH4+-N removal rate was comparable or higher in the case of the AnS cultures. Further, the AeS and AnS cultures showed significantly different lipid production characteristics in terms of the fatty acid composition and the response to nitrogen deficiency. Nitrogen starvation caused changes in the microbial community structures in all the experimental cultures, which may have influenced the lipid metabolism and the microalgal growth. The overall results suggest that both the inocula exhibit good potential with regard to the treatment of AD effluents.

Biomethanation of Harmful Macroalgal Biomass in Leach-Bed Reactor Coupled to Anaerobic Filter: Effect of Water Regime and Filter Media.

Ulva is a marine macroalgal genus which causes serious green tides in coastal areas worldwide. This study investigated anaerobic digestion as a way to manage Ulva waste in a leach-bed reactor coupled to an anaerobic filter (LBR-AF). Two LBR-AF systems with different filter media, blast furnace slag grains for R1, and polyvinyl chloride rings for R2, were run at increasing water replacement rates (WRRs). Both achieved efficient volatile solids reduction (68.4⁻87.1%) and methane yield (148⁻309 mL/g VS fed) at all WRRs, with the optimal WRR for maximum methane production being 100 mL/d. R1 maintained more stable methanation performance than R2, possibly due to the different surface properties (i.e., biomass retention capacity) of the filter media. Such an effect was also noted in the different behaviors of the LBR and AF between R1 and R2. The molecular analysis results revealed that the development of the microbial community structure in the reactors was primarily determined by the fermentation type, i.e., dry (LBR) or wet (AF).

Anaerobic co-digestion of high-strength organic wastes pretreated by thermal hydrolysis.

Thermal hydrolysis (TH) pretreatment was investigated for the anaerobic digestion (AD) of a mixture of high-strength organic wastes (i.e., dewatered human feces, dewatered sewage sludge, and food wastewater) at laboratory scale to simulate a full-scale plant and evaluate its feasibility. The reactors maintained efficient and stable performance at a hydraulic retention time of 20 days, which may be not sufficient for the mesophilic AD of high-suspended-solid wastes, despite the temporal variations in organic load. The addition of FeCl3 was effective in controlling H2S and resulted in significant changes in the microbial community structure, particularly the methanogens. The temporary interruption in feeding or temperature control led to immediate performance deterioration, but it recovered rapidly when normal operations were resumed. The overall results suggest that the AD process coupled with TH pretreatment can provide an efficient, robust, and resilient system to manage high-suspended-solid wastes, supporting the feasibility of its full-scale implementation.

A comparative study of single- and two-phase anaerobic digestion of food waste under uncontrolled pH conditions.

This study compared single- versus two-phase systems for semi-continuous anaerobic digestion of food waste without pH control at varying organic loading rates (OLRs). The methanogenic reactors of both systems required trace element supplementation for stable operation at 3.0 g VS (volatile solids)/L∙d or higher OLRs. Under trace-element supplemented conditions, both systems achieved stable and efficient performance at OLRs up to 4.0 g VS/L∙d. The two-phase system outperformed the single-phase system at 1.0-4.0 g VS/L∙d OLRs, but it failed at an OLR of 5.0 g VS/L∙d. Meanwhile, the single-phase system maintained the stable performance and reached its maximum methane production at this OLR. These results suggest that a single-phase configuration is more advantageous for robust treatment of food waste without pH control at high organic and hydraulic loads. Hydrogenotrophic methanogens dominated the methanogen community throughout the experiment in both systems. Microbial community structure shifts correlated with reactor operation and performance characteristics.

Ulva biomass as a co-substrate for stable anaerobic digestion of spent coffee grounds in continuous mode.

Ulva biomass was evaluated as a co-substrate for anaerobic digestion of spent coffee grounds at varying organic loads (0.7-1.6g chemical oxygen demand (COD)/Ld) and substrate compositions. Co-digestion with Ulva (25%, COD basis) proved beneficial for SCG biomethanation in both terms of process performance and stability. The beneficial effect is much more pronounced at higher organic and hydraulic loads, with the highest COD removal and methane yield being 51.8% and 0.19L/g COD fed, respectively. The reactor microbial community structure changed dynamically during the experiment, and a dominance shift from hydrogenotrophic to aceticlastic methanogens occurred with increase in organic loading rate. Network analysis provides a comprehensive view of the microbial interactions involved in the system and confirms a direct positive correlation between Ulva input and methane productivity. A group of populations, including Methanobacterium- and Methanoculleus-related methanogens, was identified as a possible indicator for monitoring the biomethanation performance.

A long-term study on the effect of magnetite supplementation in continuous anaerobic digestion of dairy effluent - Magnetic separation and recycling of magnetite.

Promotion of direct interspecies electron transfer (DIET) between exoelectrogenic bacteria and electron-utilizing methanogens has recently been discussed as a new method for enhanced biomethanation. This study evaluated the effect of magnetite-promoted DIET in continuous anaerobic digestion of dairy effluent and tested the magnetic separation and recycling of magnetite to avoid continuous magnetite addition. The applied magnetite recycling method effectively supported enhanced DIET activity and biomethanation performance over a long period (>250days) without adding extra magnetite. DIET via magnetite particles as electrical conduits was likely the main mechanism for the enhanced biomethanation. Magnetite formed complex aggregate structures with microbes, and magnetite recycling also helped retain more biomass in the process. Methanosaeta was likely the major methanogen group responsible for DIET-based methanogenesis, in association with Proteobacteria and Chloroflexi populations as syntrophic partners. The recycling approach proved robust and effective, highlighting the potential of magnetite recycling for high-rate biomethanation.

Anaerobic co-digestion of spent coffee grounds with different waste feedstocks for biogas production.

Proper management of spent coffee grounds has become a challenging problem as the production of this waste residue has increased rapidly worldwide. This study investigated the feasibility of the anaerobic co-digestion of spent coffee ground with various organic wastes, i.e., food waste, Ulva, waste activated sludge, and whey, for biomethanation. The effect of co-digestion was evaluated for each tested co-substrate in batch biochemical methane potential tests by varying the substrate mixing ratio. Co-digestion with waste activated sludge had an apparent negative effect on both the yield and production rate of methane. Meanwhile, the other co-substrates enhanced the reaction rate while maintaining methane production at a comparable or higher level to that of the mono-digestion of spent coffee ground. The reaction rate increased with the proportion of co-substrates without a significant loss in methanation potential. These results suggest the potential to reduce the reaction time and thus the reactor capacity without compromising methane production.

Effect of low pH start-up on continuous mixed-culture lactic acid fermentation of dairy effluent.

Mixed-culture fermentation that does not require an energy-intensive sterilization process is a viable approach for the economically feasible production of lactic acid (LA) due to the potential use of organic waste as feedstock. This study investigated mixed-culture LA fermentation of whey, a high-strength organic wastewater, in continuous mode. Variations in the hydraulic retention time (HRT) from 120 to 8 h under different pH regimes in two thermophilic reactors (55 °C) were compared for their fermentation performance. One reactor was maintained at a low pH (pH 3.0) during operation at HRTs of 120 to 24 h and then adjusted to pH 5.5 in the later phases of fermentation at HRTs of 24 to 8 h (R1), while the second reactor was maintained at pH 5.5 throughout the experiment (R2). Although the LA production in R1 was negligible at low pH, it increased dramatically after the pH was raised to 5.5 and exceeded that in R2 when stabilized at HRTs of 8 and 12 h. The maximum yield (0.62 g LA/g substrate fed as the chemical oxygen demand (COD) equivalent), the production rate (11.5 g/L day), and the selectivity (95 %) of LA were all determined at a 12-h HRT in R1. Additionally, molecular and statistical analyses revealed that changes in the HRT and the pH significantly affected the bacterial community structure and thus the fermentation characteristics of the experimental reactors. Bacillus coagulans was likely the predominant LA producer in both reactors. The overall results suggest that low pH start-up has a positive effect on yield and selectivity in mixed-culture LA fermentation.

A long-term study on the effect of magnetite supplementation in continuous anaerobic digestion of dairy effluent - Enhancement in process performance and stability.

Interspecies electron transfer (IET) between microbial populations with different functions is critical to stable anaerobic digestion. This study, in an attempt to facilitate IET, investigated the effect of magnetite supplementation on the biomethanation of dairy effluent in continuous mode. The magnetite-added reactor (RM) was significantly more resistant and resilient to process imbalance than the reactor run without magnetite addition (RC). RC showed unstable performance with repeated process upsets, but its performance improved to be comparable to that of RM after applying magnetite supplementation. Magnetite was particularly effective in stabilizing a build-up of propionic acid and therefore improving the process robustness and reliability. The enhanced biomethanation in terms of productivity and stability was attributed to the facilitated direct IET (DIET) between exoelectrogens and methanogens via magnetite particles. Methanosaeta was the predominant methanogen group in the experimental reactors and likely played a key role in both DIET-mediated carbon dioxide-reducing and aceticlastic methanogenesis.

Continuous anaerobic co-digestion of Ulva biomass and cheese whey at varying substrate mixing ratios: Different responses in two reactors with different operating regimes.

The feasibility of co-digestion of Ulva with whey was investigated at varying substrate mixing ratios in two continuous reactors run with increasing and decreasing proportions of Ulva, respectively. Co-digestion with whey proved beneficial to the biomethanation of Ulva, with the methane yield being greater by up to 1.6-fold in co-digestion phases than in the Ulva mono-digestion phases. The experimental reactors responded differently, in terms of process performance and community structure, to the changes in the substrate mixing ratio. This can be attributed to the different operating regimes between two reactors, which may have caused the microbial communities to develop in different ways to acclimate. Methanosaeta-related populations were the predominant methanogens responsible for the production of methane regardless of different substrate mixing ratios in both reactors. Considering the methane recovery and the Ulva treatment capacity, the optimal fraction of Ulva in the substrate mixture is suggested to be 50-75%.

Continuous fermentation of food waste leachate for the production of volatile fatty acids and potential as a denitrification carbon source.

This study investigated the simultaneous effects of hydraulic retention time (HRT) and pH on the continuous production of VFAs from food waste leachate using response surface analysis. The response surface approximations (R(2)=0.895, p<0.05) revealed that pH has a dominant effect on the specific VFA production (PTVFA) within the explored space (1-4-day HRT, pH 4.5-6.5). The estimated maximum PTVFA was 0.26g total VFAs/g CODf at 2.14-day HRT and pH 6.44, and the approximation was experimentally validated by running triplicate reactors under the estimated optimum conditions. The mixture of the filtrates recovered from these reactors was tested as a denitrification carbon source and demonstrated superior performance in terms of reaction rate and lag length relative to other chemicals, including acetate and methanol. The overall results provide helpful information for better design and control of continuous fermentation for producing waste-derived VFAs, an alternative carbon source for denitrification.

Mild-temperature thermochemical pretreatment of green macroalgal biomass: Effects on solubilization, methanation, and microbial community structure.

The effects of mild-temperature thermochemical pretreatments with HCl or NaOH on the solubilization and biomethanation of Ulva biomass were assessed. Within the explored region (0-0.2M HCl/NaOH, 60-90°C), both methods were effective for solubilization (about 2-fold increase in the proportion of soluble organics), particularly under high-temperature and high-chemical-dose conditions. However, increased solubilization was not translated into enhanced biogas production for both methods. Response surface analysis statistically revealed that HCl or NaOH addition enhances the solubilization degree while adversely affects the methanation. The thermal-only treatment at the upper-limit temperature (90°C) was estimated to maximize the biogas production for both methods, suggesting limited potential of HCl/NaOH treatment for enhanced Ulva biomethanation. Compared to HCl, NaOH had much stronger positive and negative effects on the solubilization and methanation, respectively. Methanosaeta was likely the dominant methanogen group in all trials. Bacterial community structure varied among the trials according primarily to HCl/NaOH addition.

Bioaugmentation of anaerobic sludge digestion with iron-reducing bacteria: process and microbial responses to variations in hydraulic retention time.

Although anaerobic digestion (AD) is a widely used option to manage waste activated sludge (WAS), there are some drawbacks related to its slow reaction rate and low energy productivity. This study examined an anaerobic WAS digester, augmented with an iron-reducing microbial consortium, relative to changes in microbial community structure and process performance at decreasing hydraulic retention times (HRTs) of 20 to 10 days. The enhanced methanation performance (approximately 40 % increase in methane yield) by the bioaugmentation was sustained until the HRT was decreased to 12.5 days, under Fe(3+)-rich conditions (ferric oxyhydroxide, 20 mM Fe). Enhanced iron-reducing activity was evidenced by the increased Fe(2+) to total Fe ratio maintained above 50 % during the stable operational phases. A further decrease in HRT to 10 days resulted in a significant performance deterioration, along with a drop in the Fe(2+) to total Fe ratio to <35 %, after four turnovers of operation. Prevailing existence of putative iron-reducing bacteria (IRBs) was identified by denaturing gradient gel electrophoresis (DGGE), with Spirochaetaceae- and Thauera-related organisms being dominant members, and clear dominance shifts among them with respect to decrease in HRT were observed. Lowering HRT led to evident shifts in bacterial community structure likely associated with washout of IRBs, leading to decreases in iron respiration activity and AD performance at a lower HRT. The bacterial community structure shifted dynamically over phases, and the community transitions correlated well with the changes in process performance. Overall, the combined biostimulation and bioaugmentation investigated in this study proved effective for enhanced methane recovery from anaerobic WAS digestion, which suggests an interesting potential for high-rate AD.

Response of a continuous anaerobic digester to temperature transitions: A critical range for restructuring the microbial community structure and function.

Temperature is a crucial factor that significantly influences the microbial activity and so the methanation performance of an anaerobic digestion (AD) process. Therefore, how to control the operating temperature for optimal activity of the microbes involved is a key to stable AD. This study examined the response of a continuous anaerobic reactor to a series of temperature shifts over a wide range of 35-65 °C using a dairy-processing byproduct as model wastewater. During the long-term experiment for approximately 16 months, the reactor was subjected to stepwise temperature increases by 5 °C at a fixed HRT of 15 days. The reactor showed stable performance within the temperature range of 35-45 °C, with the methane production rate and yield being maximum at 45 °C (18% and 26% greater, respectively, than at 35 °C). However, the subsequent increase to 50 °C induced a sudden performance deterioration with a complete cessation of methane recovery, indicating that the temperature range between 45 °C and 50 °C had a critical impact on the transition of the reactor's methanogenic activity from mesophilic to thermophilic. This serious process perturbation was associated with a severe restructuring of the reactor microbial community structure, particularly of methanogens, quantitatively as well as qualitatively. Once restored by interrupted feeding for about two months, the reactor maintained fairly stable performance under thermophilic conditions until it was upset again at 65 °C. Interestingly, in contrast to most previous reports, hydrogenotrophs largely dominated the methanogen community at mesophilic temperatures while acetotrophs emerged as a major group at thermophilic temperature. This implies that the primary methanogenesis route of the reactor shifted from hydrogen- to acetate-utilizing pathways with the temperature shifts from mesophilic to thermophilic temperatures. Our observations suggest that a mesophilic digester may not need to be cooled at up to 45 °C in case of undesired temperature rise, for example, by excessive self-heating, which offers a possibility to reduce operating costs.