Pilot-scale membrane-covered composting of food waste: Initial moisture, mature compost addition, aeration time and rate.

The impact of different operational parameters on the composting efficiency and compost quality during pilot-scale membrane-covered composting (MCC) of food waste (FW) was evaluated. Four factors were assessed in an orthogonal experiment at three different levels: initial mixture moisture (IMM, 55 %, 60 %, and 65 %), aeration time (AT, 6, 9, and 12 h/d), aeration rate (AR, 0.2, 0.4, and 0.6 m3/h) and mature compost addition ratio (MC, 2 %, 4 %, and 6 %). Results indicated that 55 % IMM, 6 h/d AT, 0.4 m3/h AR, and 4 % MC addition ratio simultaneously provided the compost with the maximum cumulative temperature and the minimum moisture. It was shown that the IMM was the driving factor of this optimum composting process. On contrary, the optimal parameters for reducing carbon and nitrogen loss were 65 % IMM, 6 h/d AT, 0.4 m3/h AR, and 2 % MC addition ratio. The AR had the most influence on reducing carbon and nitrogen losses compared to all other factors. The optimal conditions for compost maturity were 55 % IMM, 9 h/d AT, 0.2 m3/h AR, and 6 % MC addition ratio. The primary element influencing the pH and electrical conductivity values was the AR, while the germination index was influenced by IMM. Protein was the main organic matter limiting the composting efficiency. The results of this study will provide guidance for the promotion and application of food waste MCC technology, and contribute to a better understanding of the mechanisms involved in MCC for organic solid waste treatment.

Metabolic responses and microbial community changes to long chain fatty acids: Ammonia synergetic co-inhibition effect during biomethanation.

Anaerobic co-digestion is an established strategy for increasing methane production of substrates. However, substrates rich in proteins and lipids could cause a long chain fatty acids (LCFA)-ammonia synergetic co-inhibition effect. The microbial mechanisms of this co-inhibition are still unclear. The current study explored the effect of the synergetic co-inhibition on microbial community changes and prediction of metabolic enzymes to reveal the microbial mechanisms of the co-inhibition effect. The results indicated that during the synergetic co-inhibition, methanogens were mainly affected by ammonia. Decreased relative abundances of Petrimonas (82%) and Paraclostridium (67%) showed that ammonia inhibition contributed to the suppression of LCFA ß-oxidation under the synergetic co-inhibition conditions. The accumulation of more LCFA could further suppress microorganisms' activities involved in several steps of anaerobic digestion. Finally, decrease of critical enzymes' abundances confirmed the synergetic co-inhibition effect. Overall, the current study provides novel insights for the alleviation of synergetic co-inhibition during anaerobic digestion.

Osmoprotectants boost adaptation and protect methanogenic microbiome during ammonia toxicity events in continuous processes.

Different osmoprotectants were used to counteract ammonia toxicity in continuous anaerobic reactors. The anaerobic microbiome osmoadaptation process and its role to the methanogenic recovery are also assessed. Three osmoprotectants (i.e., glycine betaine, MgCl2 and KCl) were respectively introduced in continuous reactors at high ammonia levels, namely RGB, RMg, RK, while a control reactor (RCtrl) was also used. After ammonia was introduced, the RGB, RMg, RK and RCtrl suffered 39.0%, 36.6%, 39.9% and 36.2% methane production loss, respectively. Osmoprotectants addition recovered significantly methane production by up to 68.9%, 54.3% and 32.2% for RGB, RMg and RK, respectively contrary to RCtrl, where production increased only by 13.6%. The recovered methane production was maintained in RGB and RMg for at least four HRTs, even after the addition of osmoprotectants was stopped, due to the formed methanogenic microbiota by osmoadaptation process, with Methanoculleus sp. as the dominant species.

Novel bioaugmentation strategy boosted with biochar to alleviate ammonia toxicity in continuous biomethanation.

This study investigated for the first time if ammonia tolerant methanogenic consortia can be stored in gel (biogel) and used in a later time on-demand as bioaugmentation inocula, to efficiently relieve ammonia inhibition in continuous biomethanation systems. Moreover, wood biochar was assessed as a potential enhancer of the novel biogel bioaugmentation process. Three thermophilic (55 °C), continuous stirred-tank reactors (RBgel, RChar and RMix), operated at 4.5 g NH4+-N L-1 were exposed to biogel, biochar and mixture of biogel and biochar, respectively, while a fourth reactor (RCtrl) was used as control. The results showed that the methane production yields of RMix, RChar and RBgel increased by 28.6%, 20.2% and 10.7%, respectively compared to RCtrl. The highest methane yield was achieved by the synergistic interaction between biogel and biochar. Additionally, biogel stimulated a rapid recovery of Methanoculleus thermophilus sp. and syntrophic acetate oxidising bacteria populations.

Comprehensive evaluation of different strategies to recover methanogenic performance in ammonia-stressed reactors.

In this study, strategies for recovery of ammonia-stressed AD reactors were attempted, by addition of preserved bioaugmentation consortium in gel (BioG), fresh consortium in liquid medium (BioL), woodchip biochar (BW), and straw biochar (BS). In comparison to control group with ammonia, effective treatments, i.e., BioG, BioL, BW and BS raised the maximum methane production rate by 77%, 23%, 35%, and 24%, respectively. BW possibly acted as interspecies electrical conduits for Direct Electron Transfer based on conductivity and SEM analysis. BioG facilitated slow release of bioaugmentation inocula from gel into the AD system, which protected them from a direct environmental shock. According to microbial analysis, both BioG, BioL and BW resulted in increased relative abundance of Methanothermobacter thermautotrophicus; and BS induced selective raise of Methanosarcina thermophila. The increase of methanogens via these strategies led to the faster recovery of the AD process.

Feeding strategies of continuous biomethanation processes during increasing organic loading with lipids or glucose for avoiding potential inhibition.

Anaerobic co-digestion is a promising solution for nutrients balance and improvement of methane production in anaerobic digestion (AD) processes. However, the knowledge about the effects of different co-substrates in manure-based AD, and different feeding strategies, on the process performance and the methanogenic microbiome pathway, are still missing. Therefore, under harsh and slow stepwise increase of organic loading rate (OLR), by addition of lipids and carbohydrates as co-substrates in continuous reactors, this study elucidated their effect on methane production and methanogenic microbiome. The results showed that, when OLR increased by adding lipids, a severe inhibition due to accumulated long-chain fatty acids (LCFA) was observed, while no significant inhibition was obtained by addition of glucose. Additionally, the LCFA inhibition in the reactor fed with lipid was alleviated by slow stepwise feeding strategy that enriched aceticlastic Methanosarcina thermophile and Methanosaeta concilii, and hydrogenotrophic Methanobacterium methanogens.

Saline fish wastewater in biogas plants - Biomethanation toxicity and safe use.

Increasing marine land-based recirculating aquaculture systems (RAS) and stricter environmental regulations, pose new challenges to the aquaculture industry on how to treat and dispose saline fish wastewater. The fish wastewater could be incorporated into biogas reactors, but currently, the effects of salinity on the biomethanation process are poorly known. This study aimed to assess the toxicity of fish wastewater with different salinities on the biomethanation process and to propose optimum co-digestion scenarios for maximal methane potential and safe use in biogas plants. Results showed that, depending on salinity and organic content, it is possible to efficiently co-digest from 3.22 to 61.85% fish wastewater (v/v, wastewater/manure) and improve the maximum methane production rate from 2.72 to 61.85%, respectively compared to cow manure mono-digestion. Additionally, salinity was identified as the main inhibitor of biomethanation process with a half-maximal inhibitory concentration (IC50) of 4.37 g L-1, while sulphate reduction was identified as a secondary inhibitor.

Insights into Ammonia Adaptation and Methanogenic Precursor Oxidation by Genome-Centric Analysis.

Ammonia released from the degradation of protein and/or urea usually leads to suboptimal anaerobic digestion (AD) when N-rich organic waste is used. However, the insights behind the differential ammonia tolerance of anaerobic microbiomes remain an enigma. In this study, the cultivation in synthetic medium with different carbon sources (acetate, methanol, formate, and H2/CO2) shaped a common initial inoculum into four unique ammonia-tolerant syntrophic populations. Specifically, various levels of ammonia tolerance were observed: consortia fed with methanol and H2/CO2 could grow at ammonia levels up to 7.25 g NH+-N/L, whereas the other two groups (formate and acetate) only thrived at 5.25 and 4.25 g NH+-N/L, respectively. Metabolic reconstruction highlighted that this divergent microbiome might be achieved by complementary metabolisms to maximize biomethane recovery from carbon sources, thus indicating the importance of the syntrophic community in the AD of N-rich substrates. Besides, sodium/proton antiporter operon, osmoprotectant/K+ regulator, and osmoprotectant synthesis operon may function as the main drivers of adaptation to the ammonia stress. Moreover, energy from the substrate-level phosphorylation and multiple energy-converting hydrogenases (e.g., Ech and Eha) could aid methanogens to balance the energy request for anabolic activities and contribute to thriving when exposed to high ammonia levels.

Hydrogenotrophic methanogens are the key for a successful bioaugmentation to alleviate ammonia inhibition in thermophilic anaerobic digesters.

Bioaugmentation to alleviate ammonia inhibition under thermophilic anaerobic digestion has never been reported, as well as the working mechanism that allows a fast and successful bioaugmentation. Thus two bioaugmentation inocula (an enriched culture, and a mixed culture composed 50/50 by Methanoculleus thermophilus and the enriched culture) on the recovery of ammonia-inhibited thermophilic continuous reactors was assessed. The results showed that bioaugmentation improved methane yield by 11-13% and decreased the volatile fatty acids (VFA) by 45-52% compared to the control reactor (abiotic augmentation). Moreover, the importance of hydrogenotrophic methanogens to a fast and successful bioaugmentation was recognized. Specifically, the instant hydrogen partial pressure reduction by the bioaugmented hydrogenotroph created thermodynamically favourable conditions for the acetate oxidation process and consequently, the catabolism of other VFA. High-throughput sequencing results strengthened this explanation by showing that the bioaugmented M. thermophilus stimulated the growth of syntrophic acetate oxidising bacterium Thermacetogenium phaeum, immediately after bioaugmentation.

Bioaugmentation strategy for overcoming ammonia inhibition during biomethanation of a protein-rich substrate.

High ammonia levels inhibit anaerobic digestion (AD) process and bioaugmentation with ammonia tolerant methanogenic culture is proposed to alleviate ammonia inhibition. In the current study, hydrogenotrophic Methanoculleus bourgensis was bioaugmented in an ammonia-inhibited continuous reactor fed mainly with microalgae (a protein-rich biomass), at extreme ammonia levels (i.e. 11 g NH4+-N L-1). The results showed 28% increase in methane production immediately after bioaugmentation. Moreover, volatile fatty acids decreased rapidly from more than 5 g L-1 to around 1 g L-1, with a fast reduction in propionate concentration. High throughput 16s rRNA gene sequencing demonstrated that the bioaugmented M. bourgensis doubled its relative abundance after bioaugmentation. "Microbiological domino effect", triggered by the bioaugmented M. bourgensis establishing a newly efficient community, was proposed as the working mechanism of the successful bioaugmentation. Additionally, a strong aceticlastic methanogenesis was found at the end of the experiment evidenced by the dominant presence of Methanosarcina soligelidi and the low abundance of syntrophic acetate oxidising bacteria at the final period. Overall, for the first time, this study proved the positive effect of bioaugmentation on ammonia inhibition alleviation of the microalgae-dominating fed reactor, paving the way of efficient utilization of other protein-rich substrates in the future.

Biogas upgrading and biochemical production from gas fermentation: Impact of microbial community and gas composition.

The present study proposes a novel alternative method of the current biogas upgrading techniques by converting CO2 (in the biogas) into valuable chemicals (e.g., volatile fatty acids) using H2 as energy source and acetogenic mixed culture as biocatalyst. The influence of thermal treatment (90 °C) on the inhibition of the methanogenic archaea and enriching the acetogenic bacteria in different inocula (mesophilic and thermophilic) was initially tested. The most efficient inoculum that achieved the highest performance through the fermentation process was further used to define the optimum H2/CO2 gas ratio that secures maximum production yield of chemicals and maximum biogas upgrading efficiency. In addition, 16S rRNA analysis of the microbial community was conducted at the end of the experimental period to target functional microbes. The maximum biogas content (77% (v/v)) and acetate yield (72%) were achieved for 2H2:1CO2 ratio (v/v), with Moorella sp. 4 as the most dominant thermophilic acetogenic bacterium.

Acclimatization contributes to stable anaerobic digestion of organic fraction of municipal solid waste under extreme ammonia levels: Focusing on microbial community dynamics.

The organic fraction of municipal solid waste (OFMSW) is an abundant and sustainable substrate for the anaerobic digestion (AD) process, yet ammonia released during OFMSW hydrolysis could result in suboptimal biogas production. Acclimatized ammonia tolerant microorganisms offer an efficient way to alleviate ammonia inhibition during AD. This study aimed to achieve an efficient AD of OFMSW under extreme ammonia levels and elucidate the dynamics of the acclimatized microbial community. Thus, two mesophilic continuous stirred tank reactors (CSTR), fed only with OFMSW, were successfully acclimatized up to 8.5 g NH4+-N/L, and their methane yields fluctuated <10%, compared to the methane yields without ammonia addition. Microbiological analyses showed that Methanosaeta concilii and Methanosarcina soligelidi were the dominant methanogens at low and high ammonia levels, respectively. Whilst, a unique metabolic pathway shift, from aceticlastic to hydrogenotrophic methanogenesis, of M. soligelidi was identified during the acclimatization process.

16s rRNA gene sequencing and radioisotopic analysis reveal the composition of ammonia acclimatized methanogenic consortia.

Different mesophilic and thermophilic methanogenic consortia were acclimatised and enriched to extreme total ammonia (9.0 and 5.0 g NH4+-N L-1, respectively) and free ammonia (1.0 and 1.4 g NH3-N L-1, respectively) levels in this study. [2-14C] acetate radioisotopic analyses showed the dominance of aceticlastic methanogenesis in all enriched consortia. According to 16S rRNA gene sequencing result, in mesophilic consortia, methylotrophic Methanomassiliicoccus luminyensis was predominant, followed by aceticlastic Methanosarcina soligelidi. A possible scenario explaining the dominance of M. luminyensis includes the use of methylamine produced by Tissierella spp. and biomass build-up by metabolizing acetate. Nevertheless, further studies are needed to pinpoint the exact metabolic pathway of M. luminyensis. In thermophilic consortia, aceticlastic Methanosarcina thermophila was the sole dominant methanogen. Overall, results derived from this study demonstrated the efficient biomethanation ability of these ammonia-tolerant methanogenic consortia, indicating a potential application of these consortia to solve ammonia toxicity problems in future full-scale reactors.

Simultaneous biogas upgrading and biochemicals production using anaerobic bacterial mixed cultures.

A novel biological process to upgrade biogas was developed and optimised during the current study. In this process, CO2 in the biogas and externally provided H2 were fermented under mesophilic conditions to volatile fatty acids (VFAs), which are building blocks of higher-value biofuels. Meanwhile, the biogas was upgraded to biomethane (CH4 >95%), which can be used as a vehicle fuel or injected into the natural gas grid. To establish an efficient fermentative microbial platform, a thermal (at two different temperatures of 70 °C and 90 °C) and a chemical pretreatment method using 2-bromoethanesulfonate were investigated initially to inhibit methanogenesis and enrich the acetogenic bacterial inoculum. Subsequently, the effect of different H2:CO2 ratios on the efficiency of biogas upgrading and production of VFAs were further explored. The composition of the microbial community under different treatment methods and gas ratios has also been unravelled using 16S rRNA analysis. The chemical treatment of the inoculum had successfully blocked the activity of methanogens and enhanced the VFAs production, especially acetate. The chemical treatment led to a significantly better acetate production (291 mg HAc/L) compared to the thermal treatment. Based upon 16S rRNA gene sequencing, it was found that H2-utilizing methanogens were the dominant species in the thermally treated inoculum, while a significantly lower abundance of methanogens was observed in the chemically treated inoculum. The highest biogas content (96% (v/v)) and acetate production were achieved for 2H2:1CO2 ratio (v/v), with Acetoanaerobium noterae, as the dominant homoacetogenic hydrogen scavenger. Results from the present study can pave the way towards more development with respect to microorganisms and conditions for high efficient VFAs production and biogas upgrading.

Microalgal process-monitoring based on high-selectivity spectroscopy tools: status and future perspectives.

Microalgae are well known for their ability to accumulate lipids intracellularly, which can be used for biofuels and mitigate CO2 emissions. However, due to economic challenges, microalgae bioprocesses have maneuvered towards the simultaneous production of food, feed, fuel, and various high-value chemicals in a biorefinery concept. On-line and in-line monitoring of macromolecules such as lipids, proteins, carbohydrates, and high-value pigments will be more critical to maintain product quality and consistency for downstream processing in a biorefinery to maintain and valorize these markets. The main contribution of this review is to present current and prospective advances of on-line and in-line process analytical technology (PAT), with high-selectivity - the capability of monitoring several analytes simultaneously - in the interest of improving product quality, productivity, and process automation of a microalgal biorefinery. The high-selectivity PAT under consideration are mid-infrared (MIR), near-infrared (NIR), and Raman vibrational spectroscopies. The current review contains a critical assessment of these technologies in the context of recent advances in software and hardware in order to move microalgae production towards process automation through multivariate process control (MVPC) and software sensors trained on "big data". The paper will also include a comprehensive overview of off-line implementations of vibrational spectroscopy in microalgal research as it pertains to spectral interpretation and process automation to aid and motivate development.

Effect of different ammonia sources on aceticlastic and hydrogenotrophic methanogens.

Ammonium chloride (NH4Cl) was usually used as a model ammonia source to simulate ammonia inhibition during anaerobic digestion (AD) of nitrogen-rich feedstocks. However, ammonia in AD originates mainly from degradation of proteins, urea and nucleic acids, which is distinct from NH4Cl. Thus, in this study, the inhibitory effect of a "natural" ammonia source (urea) and NH4Cl, on four pure methanogenic strains (aceticlastic: Methanosarcina thermophila, Methanosarcina barkeri; hydrogenotrophic: Methanoculleus bourgensis, Methanoculleus thermophilus), was assessed under mesophilic (37 °C) and thermophilic (55 °C) conditions. The results showed that urea hydrolysis increased pH significantly to unsuitable levels for methanogenic growth, while NH4Cl had a negligible effect on pH. After adjusting initial pH to 7 and 8, urea was significantly stronger inhibitor with longer lag phases to methanogenesis compared to NH4Cl. Overall, urea seems to be more toxic on both aceticlastic and hydrogenotrophic methanogens compared to NH4Cl under the same total and free ammonia levels.

Acclimation to extremely high ammonia levels in continuous biomethanation process and the associated microbial community dynamics.

Acclimatized anaerobic communities to high ammonia levels can offer a solution to the ammonia toxicity problem in biogas reactors. In the current study, a stepwise acclimation strategy up to 10g NH4+-N L-1, was performed in mesophilic (37±1°C) continuously stirred tank reactors. The reactors were co-digesting (20/80 based on volatile solid) cattle slurry and microalgae, a protein-rich, 3rd generation biomass. Throughout the acclimation period, methane production was stable with more than 95% of the uninhibited yield. Next generation 16S rRNA gene sequencing revealed a dramatic microbiome change throughout the ammonia acclimation process. Clostridium ultunense, a syntrophic acetate oxidizing bacteria, increased significantly alongside with hydrogenotrophic methanogen Methanoculleus spp., indicating strong hydrogenotrophic methanogenic activity at extreme ammonia levels (>7g NH4+-N L-1). Overall, this study demonstrated for the first time that acclimation of methanogenic communities to extreme ammonia levels in continuous AD process is possible, by developing a specialised acclimation AD microbiome.

A systematic methodology to extend the applicability of a bioconversion model for the simulation of various co-digestion scenarios.

Detailed simulation of anaerobic digestion (AD) requires complex mathematical models and the optimization of numerous model parameters. By performing a systematic methodology and identifying parameters with the highest impact on process variables in a well-established AD model, its applicability was extended to various co-digestion scenarios. More specifically, the application of the step-by-step methodology led to the estimation of a general and reduced set of parameters, for the simulation of scenarios where either manure or wastewater were co-digested with different organic substrates. Validation of the general parameter set involved the simulation of laboratory-scale data from three continuous co-digestion experiments, treating mixtures of different organic residues either at thermophilic or mesophilic conditions. Evaluation of the results showed that simulations using the general parameter set fitted experimental data quite well, indicating that it offers a reliable reference point for future simulations of anaerobic co-digestion scenarios.

Different cultivation methods to acclimatise ammonia-tolerant methanogenic consortia.

Bioaugmentation with ammonia tolerant-methanogenic consortia was proposed as a solution to overcome ammonia inhibition during anaerobic digestion process recently. However, appropriate technology to generate ammonia tolerant methanogenic consortia is still lacking. In this study, three basic reactors (i.e. batch, fed-batch and continuous stirred-tank reactors (CSTR)) operated at mesophilic (37°C) and thermophilic (55°C) conditions were assessed, based on methane production efficiency, incubation time, TAN/FAN (total ammonium nitrogen/free ammonia nitrogen) levels and maximum methanogenic activity. Overall, fed-batch cultivation was clearly the most efficient method compared to batch and CSTR. Specifically, by saving incubation time up to 150%, fed-batch reactors were acclimatised to nearly 2-fold higher FAN levels with a 37%-153% methanogenic activity improvement, compared to batch method. Meanwhile, CSTR reactors were inhibited at lower ammonia levels. Finally, specific methanogenic activity test showed that hydrogenotrophic methanogens were more active than aceticlastic methanogens in all FAN levels above 540mgNH3-NL-1.

Ammonia tolerant inocula provide a good base for anaerobic digestion of microalgae in third generation biogas process.

This study investigated the ability of an ammonia-acclimatized inoculum to digest efficiently protein-rich microalgae for continuous 3rd generation biogas production. Moreover, we investigated whether increased C/N ratio could alleviate ammonia toxicity. The biochemical methane potential (BMP) of five different algae (Chlorella vulgaris)/manure (cattle) mixtures showed that the mixture of 80/20 (on VS basis) resulted in the highest BMP value (431mLCH4 gVS-1), while the BMP of microalgae alone (100/0) was 415mLCH4 gVS-1. Subsequently, anaerobic digestion of those two substrates was tested in continuous stirred tank reactors (CSTR). Despite of the high ammonium levels (3.7-4.2g NH4+-NL-1), CSTR reactors using ammonia tolerant inoculum resulted in relatively high methane yields (i.e. 77.5% and 84% of the maximum expected, respectively). These results demonstrated that ammonia tolerant inocula could be a promising approach to successfully digest protein-rich microalgae and achieve a 3rd generation biogas production.