Effect of Stepwise Exposure to High-Level Erythromycin on Anaerobic Digestion.

High-level erythromycin (ERY) fermentation wastewater will pose serious threats to lake environments. Anaerobic digestion (AD) has advantages in treating high-level antibiotic wastewater. However, the fate of antibiotic resistance genes (ARGs) and microbial communities in AD after stepwise exposure to high-level ERY remains unclear. In this study, an AD reactor was first exposed to 0, 5, 10, 50, 100 and 200 mg/L ERY and then re-exposed to 0, 50, 200 and 500 mg/L ERY to investigate the effect of ERY on AD. The results show that AD could adapt to the presence of high-level ERY (500 mg/L) and could maintain efficient CH4 production after domestication with low-level ERY (50 mg/L). The AD process could achieve higher removal of ERY (>94%), regardless of the initial ERY concentration. ErmB and mefA, conferring resistance through target alteration and efflux pumps, respectively, were dominant in the AD process. The first exposure to ERY stimulated an increase in the total ARG abundance, while the AD process seemed to discourage ARG maintenance following re-exposure to ERY. ERY inhibited the process of acetoclastic methanogenesis, but strengthened the process of hydrogenotrophic methanogenesis. This work provides useful information for treating high-level ERY fermentation wastewater by the AD process.

Escherichia coli-based biorefining process yields optically pure lactic acid from fermented second-generation feedstocks.

Within the circular bioeconomy the production of optically pure LA from 2nd generation feedstocks would be ideal but it is very challenging. In this paper genetically engineered Escherichia coli strains were created to resolve racemic LA solutions synthesised and produced from the fermentation of organic waste or ensiled grass. Refining LA racemic mixtures into either a D- or L-LA was achieved by cells being able to consume one LA isomer as a sole carbon and energy source while not being able to consume the other. A D-LA refining strain JSP0005 was grown on fermented source-sorted organic household waste and different grass silage leachates, which are 2nd generation feedstocks containing up to 33 g/L lactic acid racemate. In all growth experiments, L-LA was completely removed leaving D-LA as the only LA stereoisomer, i.e. resulting in optically pure D-LA, which also increased by as much as 248.6 % from its starting concentration, corresponding to 38 g/L. The strains resulting from this study are a promising first step towards a microbial based LA biorefining process.

Carbon monoxide inhibition on acidogenic glucose fermentation and aceticlastic methanogenesis.

Syngas and CO-rich off-gases are key chemical platforms to produce biofuels and bioproducts. From the perspective of optimizing and up-scaling CO co-digestion with organic waste streams, this study aims at assessing and quantifying the inhibitory effects of CO on acidogenic glucose fermentation and aceticlastic methanogenesis. Mesophilic cultures were fed in two sets of batch assays, respectively, with glucose and acetate while being exposed to dissolved CO in equilibrium with partial pressures in the range of 0.25-1.00 atm. Cumulative methane production and microbial monitoring revealed that aceticlastic methanogenic archaea were significantly inhibited (2-20 % of the methane production of CO non-exposed cultures). The acidogenic glucose degrading community was also inhibited by CO, although, thanks to its functional redundancy, shifted its metabolism towards propionate production. Future work should assess the sensitivity of hereby estimated CO inhibition parameters, e.g., on the simulation output of a continuous syngas co-digestion process with organic substrates.

A Novel Bio-Purification Process Employing an Engineered E. coli Strain for Downstream Processing of Lactic Acid Solutions from the Fermentation of Agro-Industrial by-Products.

This study aims to integrate a novel bio-purification process employing an engineered E. coli strain in the downstream processing of lactic acid (LA) fermentation broths from low-cost renewable biological feedstocks. Fermentation broth of candy waste and digestate mixture was used as a real biological feedstock. An engineered E. coli strain that selectively catabolize impurities without catabolizing LA was initially adapted on the biological feedstock, followed by shake flask experiments to prove the bio-purification concept. Scale-up and validation in a bench-scale bioreactor followed, before developing a semi-continuous membrane bioreactor (MBR) bio-purification process. The MBR bio-purification was assessed with biological feedstocks which simulated ultrafiltration or nanofiltration permeates. Incomplete removal of impurities and increased fouling was observed in the case of the ultrafiltration permeate. Contrarily, the nanofiltration permeate was successfully treated with MBR bio-purification, since low membrane fouling, 100% maltose and acetic acid removal, and no LA catabolism was achieved. MBR bio-purification as a post-treatment step in the downstream processing of LA was demonstrated as a promising technology for increasing the purity of LA solutions.

Enhanced direct gaseous CO2 fixation into higher bio-succinic acid production and selectivity.

Utilizing CO2 for bio-succinic acid production is an attractive approach to achieve carbon capture and recycling (CCR) with simultaneous production of a useful platform chemical. Actinobacillus succinogenes and Basfia succiniciproducens were selected and investigated as microbial catalysts. Firstly, the type and concentration of inorganic carbon concentration and glucose concentration were evaluated. 6 g C/L MgCO3 and 24 g C/L glucose were found to be the optimal basic operational conditions, with succinic acid production and carbon yield of over 30 g/L and over 40%, respectively. Then, for maximum gaseous CO2 fixation, carbonate was replaced with CO2 at different ratios. The "less carbonate more CO2" condition of the inorganic carbon source was set as carbonate: CO2 = 1:9 (based on the mass of carbon). This condition presented the highest availability of CO2 by well-balanced chemical reaction equilibrium and phase equilibrium, showing the best performance with regarding CO2 fixation (about 15 mg C/(L·hr)), with suppressed lactic acid accumulation. According to key enzymes analysis, the ratio of phosphoenolpyruvate carboxykinase to lactic dehydrogenase was enhanced at high ratios of gaseous CO2, which could promote glucose conversion through the succinic acid path. To further increase gaseous CO2 fixation and succinic acid production and selectivity, stepwise CO2 addition was evaluated. 50%-65% increase in inorganic carbon utilization was obtained coupled with 20%-30% increase in succinic acid selectivity. This was due to the promotion of the succinic acid branch of the glucose metabolism, while suppressing the pyruvate branch, along with the inhibition on the conversion from glucose to lactic acid.

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.

A Coculture of Photoautotrophs and Hydrolytic Heterotrophs Enables Efficient Upcycling of Starch from Wastewater toward Biomass-Derived Products: Synergistic Interactions Impacting Metabolism of the Consortium.

Even with particular interest in sustainable development, due to the limited types of bioavailable carbon sources that could support heterotrophic/mixotrophic growth, microalgae-derived products still suffer from inconsistent yield and high costs. This study demonstrates a successful cocultivation of the photoautotroph Chlorella vulgaris with a hydrolytic-enzyme-abundant heterotroph, Saccharomycopsis fibuligera, enabling efficient starch upcycling from water/wastewater toward enhancing microalgae-dominant biomass and lipid production. The enzymatic activities of S. fibuligera contributed to the hydrolysis of starch into glucose, generating a 7-fold higher biomass through mixotrophic/heterotrophic growth of C. vulgaris. Further, scanning transmission electron microscopy (STEM) and quantitative analysis suggested a significantly induced accumulation of lipids in C. vulgaris. Results of meta-transcriptomics revealed the critical regulatory role of illumination in interaction shifting. Gene expression for glycolysis and lipid biosynthesis of C. vulgaris were highly activated during dark periods. Meanwhile, during illumination periods, genes coding for glucoamylase and the sulfur-related activities in S. fibuligera were significantly upregulated, leading to induced starch hydrolysis and potential increased competition for sulfur utilization, respectively. This study indicates that hydrolytic organisms could collaborate to make starch bioavailable for nonhydrolytic microalgae, thus broadening the substrate spectrum and making starch a novel biotechnological feedstock for microalgae-derived products, e.g., biofuels or single-cell protein.

Microalgae as tools for bio-circular-green economy: Zero-waste approaches for sustainable production and biorefineries of microalgal biomass.

Microalgae are promising organisms that are rapidly gaining much attention due to their numerous advantages and applications, especially in biorefineries for various bioenergy and biochemicals. This review focuses on the microalgae contributions to Bio-Circular-Green (BCG) economy, in which zero-waste approaches for sustainable production and biorefineries of microalgal biomass are introduced and their possible integration is discussed. Firstly, overviews of wastewater upcycling and greenhouse gas capture by microalgae are given. Then, a variety of valuable products from microalgal biomass, e.g., pigments, vitamins, proteins/peptides, carbohydrates, lipids, polyunsaturated fatty acids, and exopolysaccharides, are summarized to emphasize their biorefinery potential. Techno-economic and environmental analyses have been used to evaluate sustainability of microalgal biomass production systems. Finally, key issues, future perspectives, and challenges for zero-waste microalgal biorefineries, e.g., cost-effective techniques and innovative integrations with other viable processes, are discussed. These strategies not only make microalgae-based industries commercially feasible and sustainable but also reduce environmental impacts.

Mechanistic insights into Fe3O4-modified biochar relieving inhibition from erythromycin on anaerobic digestion.

Anaerobic digestion (AD) of antibiotic manufacturing wastewater to degrade residual antibiotics and produce mixture of combustible gases has been investigated actively in the past decades. However, detrimental effect of residual antibiotic to microbial activities is commonly faced in AD process, leading to the reduction of treatment efficiency and energy recovery. Herein, the present study systematically evaluated the detoxification effect and mechanism of Fe3O4-modified biochar in AD of erythromycin manufacturing wastewater. Results showed that Fe3O4-modified biochar had stimulatory effect on AD at 0.5 g/L erythromycin existence. A maximum methane yield of 327.7 ± 8.0 mL/g COD was achieved at 3.0 g/L Fe3O4-modified biochar, leading to the increase of 55.7% compared to control group. Mechanistic investigation demonstrated that different levels of Fe3O4-modified biochar could improve methane yield via different metabolic pathways involved in specific bacteria and archaea. Low levels of Fe3O4-modified biochar (i.e., 0.5-1.0 g/L) led to the enrichment of Methanothermobacter sp., strengthening the hydrogenotrophic pathway. On the contrary, high levels of Fe3O4-modified biochar (2.0-3.0 g/L) favored the proliferation of acetogens (e.g., Lentimicrobium sp.) and methanogen (Methanosarcina sp.) and their syntrophic relations played vital role on the simulated AD performance at erythromycin stress. Additionally, the addition of Fe3O4-modified biochar significantly decreased the abundance of representative antibiotic resistant genes (ARGs), benefiting the reduction of environmental risk. The results of this study verified that the application of Fe3O4-modified biochar could be an efficient approach to detoxify erythromycin on AD system, which brings high impacts and positive implications for biological antibiotic wastewater 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.

Ozonation-pelleting of nitrogen-enriched wheat straw: Towards improved pellet properties, enhanced digestibility, and reduced methane emissions.

The livestock industry needs to use crop straws that are highly digestible to improve feed productivity and reduce ruminal methane emissions. Hence, this study aimed to use the ozonation and pelleting processes to enhance the digestibility and reduce the ruminal methane emissions of wheat straw enriched with two nitrogen sources (i.e., urea and heat-processed broiler litter). Various analyses were conducted on the pellets, including digestibility indicators, mechanical properties, surface chemistry functionalization, chemical-spectral-structural features, and energy requirements. For comparison, loose forms of the samples were also analyzed. The nitrogen-enriched ozonated wheat straw pellets had 43.06 % lower lignin, 28.30 % higher gas production for 24 h, 12.28 % higher metabolizable energy, 13.78 % higher in vitro organic matter digestibility for 24 h, and 28.81 % higher short-chain fatty acid content than the nitrogen-enriched loose sample. The reduction of methane emissions by rumen microorganisms of nitrogen-enriched wheat straw by ozonation, pelleting, and ozonation-pelleting totaled 89.15 %, 23.35 %, and 66.98 %, respectively. The ozonation process resulted in a 64 % increase in the particle density, a 5.5-time increase in the tensile strength, and a 75 % increase in the crushing energy of nitrogen-enriched wheat straw. In addition, ozone treatment could also reduce the specific and thermal energy consumption required in the pelleting process by 15.10 % and 7.61 %, respectively.

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.

Putting the electro-bugs to work: A systematic review of 22 years of advances in bio-electrochemical systems and the parameters governing their performance.

Wastewater treatment using bioelectrochemical systems (BESs) can be considered as a technology finding application in versatile areas such as for renewable energy production and simultaneous reducing environmental problems, biosensors, and bioelectrosynthesis. This review paper reports and critically discusses the challenges, and advances in bio-electrochemical studies in the 21st century. To sum and critically analyze the strides of the last 20+ years on the topic, this study first provides a comprehensive analysis on the structure, performance, and application of BESs, which include Microbial Fuel Cells (MFCs), Microbial Electrolysis Cells (MECs) and Microbial Desalination Cells (MDCs). We focus on the effect of various parameters, such as electroactive microbial community structure, electrode material, configuration of bioreactors, anode unit volume, membrane type, initial COD, co-substrates and the nature of the input wastewater in treatment process and the amount of energy and fuel production, with the purpose of showcasing the modes of operation as a guide for future studies. The results of this review show that the BES have great potential in reducing environmental pollution, purifying saltwater, and producing energy and fuel. At a larger scale, it aspires to facilitate the path of achieving sustainable development and practical application of BES in real-world scenarios.

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.

Daphnia magna as biological harvesters for green microalgae grown on recirculated aquaculture system effluents.

The sustainability of recycling aquaculture systems (RAS) is challenged by nutrient discharges, which cause water eutrophication. Efficient treatments for RAS effluents are needed to mitigate its environmental impacts. Microalgae assimilate nutrients and dissolved carbon into microbial biomass with value as feed or food ingredient. However, they are difficult to harvest efficiently. Daphnia magna is an efficient filter feeder that grazes on microalgae at high rates and serves as valuable fish feed. Combining nutrient removal by microalgae and biomass harvesting by D. magna could be a cost-effective solution for wastewater valorization. Nutrient removal from unsterilized aquaculture wastewater was evaluated using the microalgae species Chlorella vulgaris, Scenedesmus dimorphus, and Haematococcus pluvialis. The first two algae were subsequently harvested using D. magna as a grazer, while H. pluvialis failed to grow stably. All phosphorus was removed, while only 50-70 % nitrogen was recovered, indicating phosphorus limitation. Shortening the hydraulic retention time (HRT) or phosphorus dosing resulted in increased nitrogen removal. C. vulgaris cultivation was unstable at 3 days HRT or when supplied with extra phosphorus at 5 days HRT. D. magna grew on produced algae accumulating protein at 20-30 % of dry weight, with an amino acid profile favorable for use as high value fish feed. Thus, this study demonstrates the application of a two steps multitrophic process to assimilate residual nutrients into live feeds suitable for fish.

Chitosan-coated oleaginous microalgae-fungal pellets for improved bioremediation of non-sterile secondary effluent and application in carbon dioxide sequestration in bubble column photobioreactors.

Oleaginous microalga Scenedesmus sp. SPP was rapidly immobilized in oleaginous fungal pellets by their opposite-surface-charges. Microalgae-fungal (MF) pellets were more effective in bioremediation of non-sterile secondary effluent than mono-culture. The optimal hydraulic retention time for dual bioremediation in semi-continuous mode was 72 h. The MF pellets coated with 0.4 %-chitosan improved removal efficiencies of COD, total nitrogen (TN), and total phosphorus (TP) up to 96.2±0.0 %, 88.2±2.8 % and 71.5±0.7 %, respectively, likely because of better cell retention and more nutrient adsorption and assimilation. Dual bioremediation by coated MF pellets was also successfully scaled up in 30-L bubble-column photobioreactors with improved COD, TN, and TP removal efficiencies of 98.5±0.0 %, 90.2±0.0 % and 79.5±2.1 %, respectively. This system also effectively removed CO2 from simulated flue gas at 71.2±0.4 % and produced biomass with high lipid content. These results highlight the effectiveness of bio-immobilization by fungal pellets; chitosan coating; and their practical applications in bioremediation and CO2 sequestration.

Micro-aeration: an attractive strategy to facilitate anaerobic digestion.

Micro-aeration can facilitate anaerobic digestion (AD) by regulating microbial communities and promoting the growth of facultative taxa, thereby increasing methane yield and stabilizing the AD process. Additionally, micro-aeration contributes to hydrogen sulfide stripping by oxidization to produce molecular sulfur or sulfuric acid. Although micro-aeration can positively affect AD, it must be strictly regulated to maintain an overall anaerobic environment that permits anaerobic microorganisms to thrive. Even so, obligate anaerobes, especially methanogens, could suffer from oxidative stress during micro-aeration. This review describes the applications of micro-aeration in AD and examines the cutting-edge advances in how methanogens survive under oxygen stress. Moreover, barriers and corresponding solutions are proposed to move micro-aeration technology closer to application at scale.

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.

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.