A synthetic biology approach for the treatment of pollutants with microalgae.

The increase in global population and industrial development has led to a significant release of organic and inorganic pollutants into water streams, threatening human health and ecosystems. Microalgae, encompassing eukaryotic protists and prokaryotic cyanobacteria, have emerged as a sustainable and cost-effective solution for removing these pollutants and mitigating carbon emissions. Various microalgae species, such as C. vulgaris, P. tricornutum, N. oceanica, A. platensis, and C. reinhardtii, have demonstrated their ability to eliminate heavy metals, salinity, plastics, and pesticides. Synthetic biology holds the potential to enhance microalgae-based technologies by broadening the scope of treatment targets and improving pollutant removal rates. This review provides an overview of the recent advances in the synthetic biology of microalgae, focusing on genetic engineering tools to facilitate the removal of inorganic (heavy metals and salinity) and organic (pesticides and plastics) compounds. The development of these tools is crucial for enhancing pollutant removal mechanisms through gene expression manipulation, DNA introduction into cells, and the generation of mutants with altered phenotypes. Additionally, the review discusses the principles of synthetic biology tools, emphasizing the significance of genetic engineering in targeting specific metabolic pathways and creating phenotypic changes. It also explores the use of precise engineering tools, such as CRISPR/Cas9 and TALENs, to adapt genetic engineering to various microalgae species. The review concludes that there is much potential for synthetic biology based approaches for pollutant removal using microalgae, but there is a need for expansion of the tools involved, including the development of universal cloning toolkits for the efficient and rapid assembly of mutants and transgenic expression strains, and the need for adaptation of genetic engineering tools to a wider range of microalgae species.

Effect of feeding frequency on the anaerobic digestion of berry fruit waste.

On-site anaerobic digesters for small agricultural farms typically have feeding schedules that fluctuate according to farm operations. Shocks in feeding, particularly for putrescible waste can disrupt the stable operation of a digester. The effect of intermittent feeding on the anaerobic digestion of rejected raspberries was investigated in four 3L reactors operated in semicontinuous mode for 350 days at 38 °C with a hydraulic retention time of 25 days and an organic loading rate (OLR) of 1gVS/L/d. During the acclimatisation period (147 days) the organic loading was 5 feeds per week. The feeding regime of two reactors was then changed while maintaining the same OLR and HRT to one weekly feed event in one reactor and 3 equal feeds per week in another. The feeding regime did not significantly affect specific methane yield (369 ± 47 L/kgVS on average) despite very different weekly patterns in methane production. Volatile fatty acids (VFA) comprised >83 % of the organics in the effluent, while the rest included non-inhibitory concentrations of phenolic compounds (515-556 mg gallic acid/L). The microbial composition and relative abundance of predominant groups in all reactors were the archaeal genera Methanobacterium and Methanolinea and the bacterial phyla Bacteridota and Firmicutes. Increasing the OLR to 2gVS/L/d on day 238 resulted in failure of all reactors, attributed to the insufficient alkalinity to counterbalance the VFA produced, and the pH decrease below 6. Overall results suggests that optimal digestion of raspberry waste is maintained despite variations in feeding frequency, but acidification can occur with OLR changes.

Fabrication of peptide-encapsulated sodium alginate hydrogel for selective gallium adsorption.

Peptides are recognized as promising adsorbents in metal selective recovery. In this study, the designed gallium-binding peptide H6GaBP was immobilized by the polysaccharide polymer sodium alginate (SA) for gallium recovery. The synthesized H6GaBP@SA microspheres exhibited a maximum adsorption capacity of 127.4 mg/g and demonstrated high selectivity for gallium at lower pH values. The adsorption process aligned well with the pseudo-second-order equation and Langmuir model. To elucidate the adsorption mechanism, a comprehensive characterization including molecular docking, scanning electron microscope coupled with energy-dispersive X-ray spectroscopy (SEM-EDS), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and thermogravimetry analysis (TGA), were conducted. These analyses revealed that gallium ions were initially adsorbed through electrostatic interaction by H6GaBP@SA, followed by a cation exchange reaction between Ga(OH)2+ and Ca2+, as well as coordination between gallium and histidine residues on the peptide. Moreover, the H6GaBP@SA exhibited improved thermal stability compared to sole sodium alginate microspheres, and the coordination of gallium with peptides can also defer the decomposition rate of the adsorbents. Compared to other adsorbents, the peptide-encapsulated hydrogel microspheres exhibited superior gallium selectivity and improved adsorption capacity, offering an environmentally friendly option for gallium recovery.

Element distribution in electrochemically treated mine wastewater for efficient resource recovery and water treatment: A pilot study.

The feasibility of recovering major and critical elements from acid mine drainage using a pilot-scale electrochemical reactor (ECR) was investigated by assessing elements concentration and species distribution in the liquid and solid phase (sludge) in multistage tests. These were carried out at different electrical currents (18-22 amps) and thus, pH (8-12). The results showed that major metals Al, Cu and Fe were removed from the liquid phase at pH 5.9 while remaining the majority of Zn (57.2 ppm). On the other hand, at pH 7, the effluent was mainly composed of Mn (7.3 ppm). These results were confirmed by the simulation results using the PHREEQC model, which also identified the main chemical species in solution and the precipitates formed after the treatment (oxyhydroxides/sulfates/oxides). The ECR treatment led to sludges with targeted critical elements, some up to 20 times (Co, Be and Sb) higher than their earth's crustal abundance. At pH 10, rare earth elements in the sludge targeted Ce, followed by Nd and La. This study is one of the few studies carrying a detailed analysis of the behavioural distribution pattern of these elements at each pH, which opens the door for the potential of recovering these elements.

Role of the substrate on Ni inhibition in biological sulfate reduction.

In treating mine-impacted waters using sulfate-reducing bacteria (SRB), metal inhibition and substrate selection are important factors affecting the efficiency of the bioprocess. This work investigated the role of the substrate (i.e. lactate, formate, glycerol and glucose) on Ni inhibition to SRB with sulfate-reducing activity tests at initial pH 5, 7 and 9 and 100 mg/L of Ni. Results indicated that the type of substrate was a significant factor affecting Ni inhibition in SRB, which was the most negligible in the lactate system, followed by glycerol, glucose, and formate. Although less significant, Ni inhibition also varied with the pH, leading for instance, to a reduction of 77% in the sulfate reducing activity for the formate system, but only of 28% for lactate at pH 5. The added substrate also influenced the precipitation kinetics and the characteristics of the precipitates, reaching Ni precipitation extents above 95%, except for glucose (83.2%).

High-Rate Sulfate Removal Coupled to Elemental Sulfur Production in Mining Process Waters Based on Membrane-Biofilm Technology.

It is anticipated that copper mining output will significantly increase over the next 20 years because of the more intensive use of copper in electricity-related technologies such as for transport and clean power generation, leading to a significant increase in the impacts on water resources if stricter regulations and as a result cleaner mining and processing technologies are not implemented. A key concern of discarded copper production process water is sulfate. In this study we aim to transform sulfate into sulfur in real mining process water. For that, we operate a sequential 2-step membrane biofilm reactor (MBfR) system. We coupled a hydrogenotrophic MBfR (H2-MBfR) for sulfate reduction to an oxidizing MBfR (O2-MBfR) for oxidation of sulfide to elemental sulfur. A key process improvement of the H2-MBfR was online pH control, which led to stable high-rate sulfate removal not limited by biomass accumulation and with H2 supply that was on demand. The H2-MBfR easily adapted to increasing sulfate loads, but the O2-MBfR was difficult to adjust to the varying H2-MBfR outputs, requiring better coupling control. The H2-MBfR achieved high average volumetric sulfate reduction performances of 1.7-3.74 g S/m3-d at 92-97% efficiencies, comparable to current high-rate technologies, but without requiring gas recycling and recompression and by minimizing the H2 off-gassing risk. On the other hand, the O2-MBfR reached average volumetric sulfur production rates of 0.7-2.66 g S/m3-d at efficiencies of 48-78%. The O2-MBfR needs further optimization by automatizing the gas feed, evaluating the controlled removal of excess biomass and S0 particles accumulating in the biofilm, and achieving better coupling control between both reactors. Finally, an economic/sustainability evaluation shows that MBfR technology can benefit from the green production of H2 and O2 at operating costs which compare favorably with membrane filtration, without generating residual streams, and with the recovery of valuable elemental sulfur.

Bottom ash from smouldered digestate and coconut coir as an alkalinity supplement for the anaerobic digestion of fruit waste.

Anaerobic digestion (AD) of readily hydrolysed substrates such as fruit waste requires the addition of a pH buffering agent. This study evaluated the use of bottom ash from the combustion of spent coconut coir and the digestate produced from rejected berry fruit and plant waste as a buffering agent. The performance of the ash was compared with using an equivalent amount of NaHCO3 as a buffering agent. Digestions of berry fruit waste were performed in 160 mL serum vials using anaerobic wastewater sludge as an inoculum. The methane yield at the optimum levels of buffering with NaHCO3 as the buffering agent was 233 ± 12 NmL CH4/g VS. The methane yield at an equivalent addition of alkalinity as ash was significantly less, (124 ± 3 NmL CH4/g VS), but still beneficial compared to the methane yield obtained from experiments with no added alkalinity (40 ± 1 NmL CH4/g VS). Further dosages of ash in systems containing optimal level of NaHCO3 buffer also revealed a decrease in the methane yield proportionally to the added ash concentration, which is suggested to be caused by soluble concentrations of Fe in the ash supplemented systems that are higher than reported inhibitory levels.

Influence of inoculum selection on the utilisation of volatile fatty acid and glucose in sulfate reducing reactors.

The capacity of three inocula (sewer biofilm, mangrove and estuary sediment) to utilise typical fermentation products of municipal solid waste for biological sulfate reduction was investigated. Each inoculum was used in two reactors, one fed a mixture of volatile fatty acids and another fed glucose to provide a suite of fermentation products via naturally occurring fermentation. Following 228 days of reactor operation, reactors inoculated with mangrove and estuary sediments exhibited higher sulfate reducing efficiencies (80-88%) compared to the biofilm-inoculated reactors (32-49%). Minimal use of acetate and its accumulation in the biofilm-inoculated reactors pointed to the high abundance of incomplete-oxidising sulfate reducing bacteria (SRB), Desulfovibrio and Desulfobulbus (90-96% of the sulfate reducing population). Although Desulfovibrio was also prominent in reactors inoculated with mangrove and estuary sediments, Desulfobacter, a known acetoclastic sulfate reducer, emerged from trace levels in these sediment (0.01% abundance in the estuary sediments and below detection in the mangrove sediments) to comprise 14%-70% of the sulfate reducing population at the end of reactor operation.

Nickel complexation as an innovative approach for nickel-cobalt selective recovery using sulfate-reducing bacteria.

This study evaluated the differences in nickel (Ni) and cobalt (Co) solubility in the presence of sulfate reducing bacteria (SRB) to evaluate the feasibility of selective recovery of both metals from mine-impacted waters. A series of sulfate reducing activity tests with Ni, Co and both metals showed that up to 99 % Ni remained soluble despite the availability of sulfide for precipitation, while Co sulfide precipitation always occurred (over 84.5 %). The characterization of proteins in the liquid phase of the experiments revealed that some proteins were only produced in the experiments where Ni displayed higher solubility, suggesting their involvement in metal complexation. Some functions of these proteins included maintaining Ni homeostasis, acting as metalloenzymes and containing Ni-binding ligands. Desulfomicrobium baculatum, Stenotrophomonas maltophilia, and Desulfovibrio magneticus, were the main responsible species producing these proteins.

Anaerobic digestion of bean straw applying a fungal pre-treatment and using cow manure as co-substrate.

The significant amounts of agriculture residues such as bean straw (BS) in rural areas, advises its valorisation for energy recovery. The feasibility of using BS for biogas production through anaerobic digestion was assessed. Prior to this, a fungal pre-treatment to hydrolyse BS with Pleutorus ostreatus was studied at 30°C and 100 rpm in orbital incubators with 1, 10 and 30 mg fungus/g straw for 14, 21 and 28 days. Then, anaerobic digestion experiments were performed in batch with cow manure (CM) as co-substrate and pre-treated BS at ratios (g/g total solids) of 1/2, 1/3, 1/5 and 0/1. Maximum lignin (18%) and hemicellulose (44%) degradation occurred at 30 mg fungus/g straw and 28 days, along with the highest total methane yield (38 mL CH4/g VS loaded). The total amount of methane decreased when increasing CM in the experiments (701.4-191.5 mL CH4), suggesting inhibition owed to a component of CM. Self-sustained biogas production of BS occurred due to the presence of bacteria (i.e. Bacilli and Bacteroidia) and archea (i.e. Methanobacteria and Methanomicrobia). However, the usage of a full-active inoculum should be studied for higher biogas production rates.

Trace elements effect on hydrolytic stage towards biogas production of model lignocellulosic substrates.

The effect and the response of several trace elements (TE) addition to the anaerobic degradation of key compounds of lignocellulosic biomass were evaluated. Lignin, cellulose and xylose were selected as principal compounds of lignocellulosic biomass. Lignin degradation was only improved by the addition of 1000 mg Fe/L, which allowed an improvement on the methane yield coefficient of 28% compared to control. SEM images from an abiotic assay showed that this effect is more likely related with a chemical effect induced by the Fe solution, instead of an enzymatic response. Pre-treatments focused on breaking the recalcitrant structure of the lignin could be more promising than TE addition for rich lignin-content substrates. Unlike to the response observed with lignin, cellulose showed a clear effect of the TE addition on methane production rate, indicating a higher preponderance of the enzymatic activity compared to the lignin biomethanization. Experiments with xylose resulted in a strong accumulation of volatile fatty acids. TE addition should be adapted to the substrate composition given the different response of each lignocellulosic compound to the different TE addition.

Characterization of sulfate-reducing bacteria dominated surface communities during start-up of a down-flow fluidized bed reactor.

An anaerobic down-flow fluidized bed reactor was inoculated with granular sludge and started-up with sulfate containing synthetic wastewater to promote the formation of a biofilm enriched in sulfate-reducing bacteria (SRB), to produce biogenic sulfide. The start-up was done in two stages operating the reactor in batch for 45 days followed by 85 days of continuous operation. Low-density polyethylene was used as support. The biofilm formation was followed up by biochemical and electron microscopy analyses and the composition of the community was examined by 16S rDNA sequence analysis. Maximum immobilized volatile solids (1.2 g IVS/L(support)) were obtained after 14 days in batch regime. During the 85 days of continuous operation, the reactor removed up to 80% of chemical oxygen demand (COD), up to 28% of the supplied sulfate and acetate was present in the effluent. Sulfate-reducing activity determined in the biofilm with ethanol or lactate as substrate was 11.7 and 15.3 g COD/g IVS per day, respectively. These results suggested the immobilization of sulfate reducers that incompletely oxidize the substrate to acetate; the phylogenetic analysis of the cloned 16S rDNA gene sequences showed high identity to the genus Desulfovibrio that oxidizes the substrates incompletely. In contrast, in the granular sludge used as inoculum a considerable number of clones showed homology to Methanobacterium and just few clones were close to SRB. The starting-up approach allowed the enrichment of SRB within the diverse community developed over the polyethylene support.