Magnetite Alters the Metabolic Interaction between Methanogens and Sulfate-Reducing Bacteria.

It is known that the presence of sulfate decreases the methane yield in the anaerobic digestion systems. Sulfate-reducing bacteria can convert sulfate to hydrogen sulfide competing with methanogens for substrates such as H2 and acetate. The present work aims to elucidate the microbial interactions in biogas production and assess the effectiveness of electron-conductive materials in restoring methane production after exposure to high sulfate concentrations. The addition of magnetite led to a higher methane content in the biogas and a sharp decrease in the level of hydrogen sulfide, indicating its beneficial effects. Furthermore, the rate of volatile fatty acid consumption increased, especially for butyrate, propionate, and acetate. Genome-centric metagenomics was performed to explore the main microbial interactions. The interaction between methanogens and sulfate-reducing bacteria was found to be both competitive and cooperative, depending on the methanogenic class. Microbial species assigned to the Methanosarcina genus increased in relative abundance after magnetite addition together with the butyrate oxidizing syntrophic partners, in particular belonging to the Syntrophomonas genus. Additionally, Ruminococcus sp. DTU98 and other species assigned to the Chloroflexi phylum were positively correlated to the presence of sulfate-reducing bacteria, suggesting DIET-based interactions. In conclusion, this study provides new insights into the application of magnetite to enhance the anaerobic digestion performance by removing hydrogen sulfide, fostering DIET-based syntrophic microbial interactions, and unraveling the intricate interplay of competitive and cooperative interactions between methanogens and sulfate-reducing bacteria, influenced by the specific methanogenic group.

Saccharina latissima, candy-factory waste, and digestate from full-scale biogas plant as alternative carbohydrate and nutrient sources for lactic acid production.

To substitute petroleum-based materials with bio-based alternatives, microbial fermentation combined with inexpensive biomass is suggested. In this study Saccharina latissima hydrolysate, candy-factory waste, and digestate from full-scale biogas plant were explored as substrates for lactic acid production. The lactic acid bacteria Enterococcus faecium, Lactobacillus plantarum, and Pediococcus pentosaceus were tested as starter cultures. Sugars released from seaweed hydrolysate and candy-waste were successfully utilized by the studied bacterial strains. Additionally, seaweed hydrolysate and digestate served as nutrient supplements supporting microbial fermentation. According to the highest achieved relative lactic acid production, a scaled-up co-fermentation of candy-waste and digestate was performed. Lactic acid reached a concentration of 65.65 g/L, with 61.69% relative lactic acid production, and 1.37 g/L/hour productivity. The findings indicate that lactic acid can be successfully produced from low-cost industrial residues.

Adaptive laboratory evolution to hypersaline conditions of lactic acid bacteria isolated from seaweed.

Seaweed biomass has been proposed as a promising alternative carbon source for fermentation processes using microbial factories. However, the high salinity content of seaweed biomass is a limiting factor in large scale fermentation processes. To address this shortcoming, three bacterial species (Pediococcus pentosaceus, Lactobacillus plantarum, and Enterococcus faecium) were isolated from seaweed biomass and evolved to increasing concentrations of NaCl. Following the evolution period, P. pentosaceus reached a plateau at the initial NaCl concentration, whereas L. plantarum, and E. faecium showed a 1.29 and 1.75-fold increase in their salt tolerance, respectively. The impact that salt evolution had on lactic acid production using hypersaline seaweed hydrolysate was investigated. Salinity evolved L. plantarum produced 1.18-fold more lactic acid than the wild type, and salinity evolved E. faecium was able to produce lactic acid, while the wild type could not. No differences in lactic acid production were observed between the P. pentosaceus salinity evolved and wild type strains. Evolved lineages were analyzed for the molecular mechanisms underlying the observed phenotypes. Mutations were observed in genes affecting the ion balance in the cell, the composition of the cell membrane and proteins acting as regulators. This study demonstrates that bacterial isolates from saline niches are promising microbial factories for the fermentation of saline substrates, without the requirement of previous desalination steps, while preserving high final product yields.

Unraveling prevalence of homoacetogenesis and methanogenesis pathways due to inhibitors addition.

Three inhibitors targeting different microorganisms, both from Archaea and Bacteria domains, were evaluated for their effect on CO2 biomethanation: sodium ionophore III (ETH2120), carbon monoxide (CO), and sodium 2-bromoethanesulfonate (BES). This study examines how these compounds affect the anaerobic digestion microbiome in a biogas upgrading process. While archaea were observed in all experiments, methane was produced only when adding ETH2120 or CO, not when adding BES, suggesting archaea were in an inactivated state. Methane was produced mainly via methylotrophic methanogenesis from methylamines. Acetate was produced at all conditions, but a slight reduction on acetate production (along with an enhancement on CH4 production) was observed when applying 20 kPa of CO. Effects on CO2 biomethanation were difficult to observe since the inoculum used was from a real biogas upgrading reactor, being this a complex environmental sample. Nevertheless, it must be mentioned that all compounds had effects on the microbial community composition.

Pilot-scale study of biomethanation in biological trickle bed reactors converting impure CO2 from a Full-scale biogas plant.

Research within biological methanation has been a great development using biotrickling filters (BTF), as a power-to-x solution, but research within up scaling is missing. This study investigates the commercial potential of biomethanation in BTF by operating two 1 m3 reactors which was implemented into a full-scale biogas plant. Several areas were investigated, such as enrichment and start-up, long-term steady state operation, serial operation, and intermittent feed. A methane productivity of [Formula: see text] with a product gas of 95.7 % CH4 was obtained for parallel operation, whereas during serial operation a methane productivity of [Formula: see text] at 97.4 % CH4 was achieved. The flexibility of the biomethanation was demonstrated with unintentional loss of H2 feed in periods of 12 to 72 h, where initial performance was regained within 6 to 12 h. The results from this study demonstrate the potential for commercial use of biomethanation in BTF for future Power-to-X solutions.

Impact of recovered phosphorus supply on methanotrophic cultivation and microbial protein production.

Microbial protein is a promising dietary supplement alternative to traditional sources, being methane oxidising bacteria (MOB) an attractive option to produce it. Though current production processes rely on fossil resources, there is an increasing trend of using recovered residual nutrient streams, with most research focusing on nitrogen and methane, paying little attention to phosphorus. Struvite and precipitated calcium phosphate (PCP) were evaluated as potential residual P sources for microbial protein production after dissolved them with strong acids. MOB growth was studied in batch experiments. Yields ranged from 0.21 to 0.29 g CDW g CH4-1. Crude protein contents above 50% of dried weight were achieved, and neither the P nor the N source affected the amino acid profile significantly. The highest protein content (75%) was observed when using struvite as nutrient source, but also yielded cadmium and lead accumulation above limits set in legislation.

Effect of pressure on biomethanation process and spatial stratification of microbial communities in trickle bed reactors under decreasing gas retention time.

The current study investigated the effect of elevating gas pressure on biomethanation in trickle-bed reactors (TBRs). The increased pressure led to successful biomethanation (CH4 > 90 %) at a gas retention time (GRT) of 21 min, due to the improved transfer rates of H2 and CO2. On the contrary, the non-pressurized TBR performance was reduced at GRTs shorter than 40 min. Metagenomic analysis revealed that the microbial populations collected from the lower and middle parts of the reactor under the same GRT were more homogeneous compared with those developed in the upper layer. Comparison with previous experiments suggest that microbial stratification is mainly driven by the nutrient provision strategy. Methanobacterium species was the most dominant methanogen and it was mainly associated with the bottom and middle parts of TBRs. Overall, the increased pressure did not affect markedly the microbial composition, while the GRT was the most important parameter shaping the microbiomes.

Untargeted Metabolomics Profiling of Bioactive Compounds under Varying Digestate Storage Conditions: Assessment of Antioxidant and Antifungal Activity.

The rapid development of biogas plants in China has generated large quantities of digestate. The disparity between the continuity of biogas plant operation and the seasonality of digestate utilization has led to the need to store digestate. Therefore, untargeted profiling of bioactive compounds in the digestate stored under aerobic and anaerobic conditions was performed. The antioxidant and antifungal activity of digestate stored under varying conditions was likewise assessed. The results delineated that digestate storage under varying conditions brought about the degradation of organic acids, alkenes, aldehydes, alcohols, ketones, ethers, amino acids and their derivatives, and esters, leading to the stabilization of digestate components. Together, these new data revealed that digestate storage for up to 20 days under aerobic conditions promotes glycine, serine, and threonine degradation pathways and enhances biotin and vitamins production. In contrast, anaerobic storage enhances the taurine and hypotaurine metabolic pathways and increases the derivation of antimicrobial substances, such as indole alkaloids. Moreover, digestate storage under anaerobic conditions promotes antioxidant and antifungal activity more than storage under aerobic conditions. These findings can contribute to the future development of high-value agricultural products from digestate and the sustainability of biogas plants. Further studies are required for the untargeted metabolomic of digestate under storage to explore the underlying mechanisms of promoting disease resistance by the digestate upon land application.

Going beyond conventional wastewater treatment plants within circular bioeconomy concept - a sustainability assessment study.

Wastewater treatment plants (WWTP) have extensive energy processes that undermine their economic and environmental performance. In this context, the integration of wastewater treatment with other biochemical processes such as co-digestion of sludge with organic wastes, and production of value-added products at their downstream processes will shift conventional WWTPs into biorefinery platforms with better sustainability performance. The sustainability of such a biorefinery platform has been investigated herein using an economic and life cycle assessment approach. This WWTP-based biorefinery treats wastewater from Copenhagen municipality, co-digests the source-sorted organic fraction of municipal solid waste and sludge, and upgrades biogas into biomethane using a hydrogen-assisted upgrading method. Apart from bioenergy, this biorefinery also produces microbial protein (MP) using recovered nutrients from WWTP's reject water. The net environmental savings achieved in two damage categories, i.e., -1.07 × 10-2 species.yr/FU in ecosystem quality and -1.68 × 106 USD/FU in resource scarcity damage categories along with high potential windows for the further environmental profile improvements make this biorefinery platform so encouraging. Despite being promising in terms of environmental performance, the high capital expenditure and low gross profit have undermined the economic performance of the proposed biorefinery. Technological improvements, process optimization, and encouraging incentives/subsidies are still needed to make this platform economically feasible.

Ex-situ biogas upgrading in thermophilic trickle bed reactors packed with micro-porous packing materials.

Two thermophilic trickle bed reactors (TBRs) were packed with different packing densities with polyurethane foam (PUF) and their performance under different retention times were evaluated during ex-situ biogas upgrading process. The results showed that the TBR more tightly packed i.e. containing more layers of PUF achieved higher H2 utilization efficiency (>99%) and thus, higher methane content (>95%) in the output gas. The tightly packed micro-porous PUF enhanced biofilm immobilization, gas-liquid mass transfer and biomethanation efficiency. Moreover, applying a continuous high-rate nutrient trickling could lead to liquid overflow resulting in formation of non-homogenous biofilm and severe deduction of biomethanation efficiency. High-throughput 16S rRNA gene sequencing revealed that the liquid media were predominated by hydrogenotrophic methanogens. Moreover, members of Peptococcaceae family and uncultured members of Clostridia class were identified as the most abundant species in the biofilm. The proliferation of hydrogenotrophic methanogens together with syntrophic bacteria showed that H2 addition resulted in altering the microbial community in biogas upgrading process.

In-situ biogas upgrading assisted by bioaugmentation with hydrogenotrophic methanogens during mesophilic and thermophilic co-digestion.

In this study, the effects of bioaugmentation of typically dominant hydrogenotrophic methanogens to CSTR co-digesting cheese whey and manure, under in-situ biomethanation operations were investigated. Reactors working at mesophilic (37 °C) and thermophilic (55 °C) conditions were independently treated and examined in terms of microbial composition and process dynamics. Addition of Methanoculleus bourgensis in the mesophilic reactor led to a stable biomethanation, and an improved microbial metabolism, resulting in 11% increase in CH4 production rate. 16S rRNA and biochemical analyses revealed an enrichment in syntrophic and acidogenic species abundance. Moreover, nearly total volatile fatty acids conversion was observed. Differently, Methanothermobacter thermautotrophicus addition in the thermophilic reactor did not promote biogas upgrading performance due to incomplete H2 conversion and inefficient community adaptation to H2 excess, ultimately favoring acetoclastic methanogenesis. Bioaugmentation constitutes a viable tool to strengthen in-situ upgrading processes and paves the way to the development of more sophisticated and robust microbial inoculants.

Enhanced fermentative lactic acid production from source-sorted organic household waste: Focusing on low-pH microbial adaptation and bio-augmentation strategy.

Lactic acid (LA) production at low pH could significantly reduce the need for neutralizing agents, leading to reduction of operational costs. In the present study, LA production at acidic conditions was investigated using source-sorted organic household waste (SSOHW). Controlling the pH at low value (i.e. 5.0) and bio-augmenting with Pediococcus acidilactici led to a concentration of 39.3 ± 0.5 g-LA/L with a yield of 0.75 ± 0.02 g-LA/g-sugar. In contrast, secondary fermentation at higher pH level (i.e. 5.5 and 6.0) resulted in complete LA degradation. Subsequently, consecutive batch fermentations were conducted to adapt P. acidilactici to SSOHW and improve the LA production. Results showed that P. acidilactici could successively adapt in the SSOHW reaching a relative abundance above 2.8% at adaptation process. The added P. acidilactici ensured a high concentration of LA at three consecutive generations, achieving an increment above 18% compared to control test (abiotic augmentation). Moreover, adaptation processes (i.e. maintaining pH at 4.0 or stepwise decreasing the pH from 5.0 to 4.0) significantly improved LA concentration and productivity at the pH of 4.0. Overall, the results provide a promising method to reduce the LA production costs using residual resources.

Valorization of municipal organic waste into purified lactic acid.

Municipal organic waste (biowaste) consists of food derived starch, protein and sugars, and lignocellulose derived cellulose, hemicellulose, lignin and pectin. Proper management enables nutrient recycling and sustainable production of platform chemicals such as lactic acid (LA). This review gathers the most important information regarding use of biowaste for LA fermentation covering pre-treatment, enzymatic hydrolysis, fermentation and downstream processing to achieve high purity LA. The optimal approach was found to treat the two biowaste fractions separately due to different pre-treatment and enzyme needs for achieving enzymatic hydrolysis and to do continues fermentation to achieve high cell density and high LA productivity up to 12 g/L/h for production of both L and D isomers. The specific productivity was 0.4 to 0.5 h-1 but with recalcitrant biomass, the enzymatic hydrolysis was rate limiting. Novel purification approaches included reactive distillation and emulsion liquid membrane separation yielding purities sufficient for polylactic acid production.

Ex-situ biogas upgrading in thermophilic up-flow reactors: The effect of different gas diffusers and gas retention times.

Four different types of ceramic gas distributors (Al2O3 of 1.2 µm and SiC of 0.5, 7 and 14 µm) were evaluated to increase biomethane formation during ex-situ biogas upgrading process. Each type of gas diffuser was tested independently at three different gas retention times of 10, 5 and 2.5 h, at thermophilic conditions. CH4 production rate increased by increasing input gas flow rate for all type of distributors, whereas CH4 concentration declined. Reactors equipped with SiC gas distributors effectively improved biomethane content fulfilling natural gas standards. Microbial analysis showed high abundance of hydrogenotrophic methanogens and proliferated syntrophic bacteria, i.e. syntrophic acetate oxidizers and homoacetogens, confirming the effect of H2 to alternate anaerobic digestion microbiome and enhance hydrogenotrophic methanogenesis. A detailed anaerobic bioconversion model was adapted to simulate the operation of the R1-R4 reactors. The model was shown to be effective for the simulation of biogas upgrading process in up-flow reactors.

Bio-electrochemically extracted nitrogen from residual resources for microbial protein production.

Upcycling of nutrients from residual resources for producing microbial protein (MP) is an attractive method to valorize residues. In this study, we investigated bio-electrochemical methods to recover ammonia-N, for further production of MP. Reject water and digestate were used for ammonia-N recovery in microbial fuel cell (MFC) system. In one-stage process, ammonia-N recovery was 32 - 42% with 57 - 154 kJ/m3 waste stream of electricity generation. For further enhancing recovery efficiency, a two-stage process was developed, achieving efficiency of 53 - 61%. Subsequently, MP was grown with the extracted ammonia-N, and amino acid concentration was 421 and 272 mg/L under 25 °C and 35 °C, respectively. Similar essential amino acid content of MP (especially under 25 °C) with the one from fish demonstrated the attractiveness of upcycling residues to proteins. Based on simplified economic evaluation, the produced energy performed the potential to catch 1.63 - 6.54 €/m3 waste stream.

Multicomponent nanoparticles as means to improve anaerobic digestion performance.

Sufficient quantity of trace metals is essential for a well performing anaerobic digestion (AD) process. Among the essential trace elements in active sites of multiple important enzymes for AD are iron and nickel ions. In the present study, iron and nickel in the form of Fe2O3 and NiO were coated on TiO2 nanoparticles to be used in batch and continuous operation mode. The effect of TiO2, Fe2O3-TiO2, and NiO-TiO2 nanoparticles on each step of AD process was assessed utilizing simple substrates (i.e. cellulose, glucose, acetic acid, and mixture of H2-CO2) as well as complex ones (i.e. municipal biopulp). The hydrolysis rate of cellulose substrate increased with higher dosages of the coated TiO2 with both metals. For instance, the hydrolysis rate was increased up to 54% at Fe2O3-TiO2 and at a concentration of 23.5 mg/L for NiO-TiO2 it was increased up to 58%, while higher dosage suppressed the hydrolytic activity. Experimental results revealed that low dosages of NiO-TiO2 increased the accumulated methane production up to 24% probably by increasing the enzymatic activity of acetoclastic methanogenesis. NiO-TiO2 showed positive effect on batch and continuous AD of biopulp and improved methane yield up to 8%.

Impact of storage duration and micro-aerobic conditions on lactic acid production from food waste.

Food waste (FW) is an abundant resource with great potential for lactic acid (LA) production. In the present study, the effect of storage time on FW characteristics and its potential for LA production was investigated. The largest part of sugars was consumed during 7 to 15 days of FW storage and the sugar consumption reached 68.0% after 15 days. To enhance the LA production, micro-aerobic conditions (13 mL air/g VS) and addition of ß-glucosidase were applied to improve polysaccharides hydrolysis, resulting to increase of monosaccharides content to 76.6%. Regarding fermentative LA production, the highest LA titer and yield of hydrolyzed FW was 32.1 ± 0.5 g/L and 0.76 ± 0.01 g/g-sugar, respectively. Furthermore, L-LA isomer was higher than 70% when FW was stored for up to 7 days. However, attention should be paid on controlling the FW storage to approximately one week.

Bioconversion of wastewater to single cell protein by methanotrophic bacteria.

Single cell protein (SCP) provides an alternative protein source to partially replace the conventional agricultural resources and support the increased nutritional needs. Inexpensive feeding source is one of the key limiting factors for the expansion of SCP production. The present study examined the valorization of biogas derived from the anaerobic digestion (AD) of sewage sludge and the discarded effluent as nutrients source to produce SCP using methanotrophic bacteria. Results indicated that the mixed methanotrophic culture can grow well on the pasteurized AD supernatant and biogas, succeeding in promising dry weight (DW) yield (0.66 ± 0.01 g-DW/g-CH4 and 11.54 ± 0.12 g-DW/g-NH4+). Methylomonas (56.26%) and Methylophilus (24.60%) spp. were the two main representatives of the mixed culture. The produced dried biomass had a protein content higher than 41% w/w, including essential amino acids like histidine, valine, phenylalanine, isoleucine, leucine, threonine and lysine. The cultivated SCP shows potential utilization as protein source for animal diets.

Potassium inhibition during sludge and biopulp co-digestion; experimental and model-based approaches.

Process instability with consecutive low methane production are common challenges of the anaerobic digestion (AD) of municipal wastes. In the present study, the co-digestion of sewage sludge and municipal biopulp was investigated at batch and continuously fed digesters. At batch tests, the highest methane yield for co-digestion (467 ± 17 mLCH4/gVS) was achieved when biopulp contributed to 80% of organic matter content and sludge the remaining 20%. At continuous mode operation, co-digestion achieved 0.91 ± 0.11 L/(L·d) methane productivity, while mono-digestion of sludge achieved 0.62 ± 0.05 L/(L·d). Potassium inhibition was investigated at the most efficient co-digestion scenario and was found that the half maximal inhibitory concentration (IC50) occurred at 8 g-K+/L. Subsequently, the effect of K+ was investigated at different scenarios at continuous operation. Simulations based on BioModel described the inhibitory effect of K+ by introducing non-competitive inhibition of methanogens. Simulation results confirmed the strongly inhibitory effect of potassium to the AD process.