A two-stage design enhanced biodegradation of high concentrations of a C16-alkyl quaternary ammonium compound in oxygen-based membrane biofilm reactors.

Quaternary ammonia compounds (QAC), such as hexadecyltrimethyl-ammonium (CTAB), are widely used as disinfectants and in personal-care products. Their use as disinfectants grew during the SARS-CoV-2 (COVID-19) pandemic, leading to increased loads to wastewater treatment systems and the environment. Though low concentrations of CTAB are biodegradable, high concentrations are toxic to bacteria. Sufficient O2 delivery is a key to achieve high CTAB removal, and the O2-based Membrane Biofilm Reactor (O2-MBfR) is a proven means to biodegrade CTAB in a bubble-free, non-foaming manner. A strategy for achieving complete biodegradation of high-concentrations of CTAB is a two-stage O2-MBfR, in which partial CTAB removal in the Lead reactor relieves inhibition in the Lag reactor. Here, more than 98 % removal of 728 mg/L CTAB could be achieved in the two-stage MBfR, and the CTAB-removal rate was 70 % higher than for a one-stage MBfR with the same O2-delivery capacity. CTAB exposure shifted the bacterial community toward Pseudomonas and Stenotrophomonas as the dominant genera. In particular, P. alcaligenes and P. aeruginosa were enriched in the Lag reactor, as they were capable of biodegrading the metabolites of initial CTAB monooxygenation. Metagenomic analysis also revealed that the Lag reactor was enriched in genes for CTAB and metabolite oxygenation, due to reduced CTAB inhibition.

Revealing the role of phosphorus supply on the phosphorus distribution and lipid production in Scenedesmus obliquus UTEX 393 during nitrogen starvation.

Microalgal-based processes offer promise for addressing two sustainability challenges: recovering phosphorus (P) from wastewater and producing biofuel feedstock. This study investigated the role of phosphorus supply on microalgal growth, lipid yield, and P distribution for Scenedesmus during nitrogen starvation. Extracellular polymeric substances and intracellular polymeric substances were the most important pools for inorganic phosphorus (IP) and organic phosphorus (OP), respectively. The main P pool for microalgae with low phosphorus supply was EPS, which accounted for 57 % of the total biomass phosphorus; while under high P concentrations, 79 % of the phosphorus was stored in IPS. A high concentration of orthophosphate stimulated rapid P uptake as IP and promoted the transformation of IP to OP associating with biomass synthesis. The highest P content of microalgal biomass was 6.5 % of dry weight when the phosphorus concentration in medium was 113 mg/L, and the OP content was 4.9 % of dry weight. High phosphate-P enhanced the biomass's lipid content by 60 %, and the distribution of fatty acid methyl esters was not altered by P concentrations. Collectively, high phosphate-P availability could promote microalgal biomass synthesis, lipid production and P accumulation.

Achieving superior carbon transfer efficiency and pH control using membrane carbonation with a wide range of CO2 contents for the coccolithophore Emiliania huxleyi.

The economic viability of microalgal-derived products relies on rapid CO2 transfer in a cost-effective manner. Many industrial gas streams contain concentrated CO2 that, if converted to useful products, would lower greenhouse gas emissions and valorize the wasted CO2. Membrane carbonation (MC) uses non-porous hollow-fiber gas-transfer membranes to deliver CO2 without bubble formation, which makes it possible to achieve a high carbon-transfer efficiency (CTE). However, inert gasses in the industrial streams (e.g., N2, O2, and H2O) can significantly lower the CO2-delivery rate. The means to overcome the buildup of inert gases in the membrane lumen is to manage the distal end of the membranes to sweep out inert gases while not wasting significant CO2. A MC-venting strategy was evaluated for CO2 inputs from 5% to 100%. Abiotic tests using a restricted exit flow could achieve >95% CTEabiotic for industrial CO2 streams. When integrated with semi-continuous cultivation of a marine coccolithophore, Emiliania huxleyi, CO2 delivery and venting were on-demand based on a pH set points and pH-actuated feed and venting valves. MC using the venting strategy achieved 100% CTEbiotic when delivering 100% and 50% CO2, which was better than 50% CTEbiotic obtained from pH-controlled sparging of 100% CO2-sparging. E. huxleyi consistently fixed ~80% of the delivered CO2 into biomass, and the remaining ~20% to calcite coccoliths. The compact size of MC modules, stable pH control, and no shear forces from bubble agitation during the CO2 delivery made MC an ideal match for cultivation of coccolithophores, which are sensitive to shear forces and pH fluctuations.

Hydrodefluorination of Perfluorooctanoic Acid in the H2-Based Membrane Catalyst-Film Reactor with Platinum Group Metal Nanoparticles: Pathways and Optimal Conditions.

PFAAs (perfluorinated alkyl acids) have become a concern because of their widespread pollution and persistence. A previous study introduced a novel approach for removing and hydrodefluorinating perfluorooctanoic acid (PFOA) using palladium nanoparticles (Pd0NPs) in situ synthesized on H2-gas-transfer membranes. This work focuses on the products, pathways, and optimal catalyst conditions. Kinetic tests tracking PFOA removal, F- release, and hydrodefluorination intermediates documented that PFOA was hydrodefluorinated by a mixture of parallel and stepwise reactions on the Pd0NP surfaces. Slow desorption of defluorination products lowered the catalyst's activity for hydrodefluorination. Of the platinum group metals studied, Pd was overall superior to Pt, Rh, and Ru for hydrodefluorinating PFOA. pH had a strong influence on performance: PFOA was more strongly adsorbed at higher pH, but lower pH promoted defluorination. A membrane catalyst-film reactor (MCfR), containing an optimum loading of 1.2 g/m2 Pd0 for a total Pd amount of 22 mg, removed 3 mg/L PFOA during continuous flow for 90 days, and the removal flux was as high as 4 mg PFOA/m2/d at a steady state. The EPA health advisory level (70 ng/L) also was achieved over the 90 days with the influent PFOA at an environmentally relevant concentration of 500 ng/L. The results document a sustainable catalytic method for the detoxification of PFOA-contaminated water.

Adsorption and Reductive Defluorination of Perfluorooctanoic Acid over Palladium Nanoparticles.

Per- and polyfluoroalkyl substances (PFASs) comprise a group of widespread and recalcitrant contaminants that are attracting increasing concern due to their persistence and adverse health effects. This study evaluated removal of one of the most prevalent PFAS, perfluorooctanoic acid (PFOA), in H2-based membrane catalyst-film reactors (H2-MCfRs) coated with palladium nanoparticles (Pd0NPs). Batch tests documented that Pd0NPs catalyzed hydrodefluorination of PFOA to partially fluorinated and nonfluorinated octanoic acids; the first-order rate constant for PFOA removal was 0.030 h-1, and a maximum defluorination rate was 16 µM/h in our bench-scale MCfR. Continuous-flow tests achieved stable long-term depletion of PFOA to below the EPA health advisory level (70 ng/L) for up to 70 days without catalyst loss or deactivation. Two distinct mechanisms for Pd0-based PFOA removal were identified based on insights from experimental results and density functional theory (DFT) calculations: (1) nonreactive chemisorption of PFOA in a perpendicular orientation on empty metallic surface sites and (2) reactive defluorination promoted by physiosorption of PFOA in a parallel orientation above surface sites populated with activated hydrogen atoms (Hads*). Pd0-based catalytic reduction chemistry and continuous-flow treatment may be broadly applicable to the ambient-temperature destruction of other PFAS compounds.

Increased expression of antibiotic-resistance genes in biofilm communities upon exposure to cetyltrimethylammonium bromide (CTAB) and other stress conditions.

Quaternary ammonium compounds (QAC, e.g., cetyltrimethylammonium bromide, (CTAB)) are widely used as surfactants and disinfectants. QAC already are commonly found in wastewaters, and their concentration could increase, since QAC are recommended to inactivate the SARS-CoV-2 (COVID-19) virus. Exposure of bacteria to QAC can lead to proliferation of antibiotic resistance genes (ARG). In particular, O2-based membrane biofilm reactors (O2-MBfRs) achieved excellent CTAB biodegradation, but ARG increased in their biofilms. Here, we applied meta-transcriptomic analyses to assess the impacts of CTAB exposure and operating conditions on microbial community's composition and ARG expression in the O2-MBfRs. Two opportunistic pathogens, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, dominated the microbial communities and were associated with the presence of ARG. Operating conditions that imposed stress on the biofilms, i.e., limited supplies of O2 and nitrogen or a high loading of CTAB, led to large increases in ARG expression, particularly for genes conferring antibiotic-target protection. Important within the efflux pumps was the Resistance-Nodulation-Division (RND) family, which may have been active in exporting CTAB from cells. Oxidative stress appeared to be the key factor that triggered ARG proliferation by selecting intrinsically resistant species and accentuating the expression of ARG. Our findings suggest that means to mitigate the spread of ARG, such as shown here in a O2-based membrane biofilm reactor, need to consider the impacts of stressors, including QAC exposure and stressful operating conditions.

Evaluation of co-culturing a diatom and a coccolithophore using different silicate concentrations.

Globally, the demand for sustainable energy production and high-value biological compounds have become intertwined in an attempt to improve the feasibility of sustainable algal cultivation. Marine microalgae, especially diatoms and coccolithophores, represent viable cultures that can produce biofuels and high-value compounds. Growing them in co-culture offers the potential to produce lipids and pigments, while also generating CaCO3 for C sequestration. The main objective of this work was to investigate competition or co-existence of the diatom Chaetoceros gracilis and the coccolithophore Pleurochrysis Carterae. The focus was on the effects of silicate and co-culturing on the growth rate, productivity, pigment production, and ash production for C. gracilis and P. carterae in laboratory conditions. The results showed that, in monoculture, 2-mM Si enhanced the specific growth rate of C. gracilis, but did not affect P. carterae. Regardless of silicate concentration, C. gracilis was more productive than P. carterae. In co-culture, P. carterae had a slower growth rate, indicating an inhibitory effect of C. gracilis on P. carterae. Neither silicate concentration nor co-culturing had an impact on the contents of pigments fucoxanthin, chlorophyll-a, and chlorophyll-c, which means that pigment productivity was proportional to biomass productivity. Finally, the ash content increased in all cultures with the lower silicate concentration (0.2 mM) in the medium. With one exception, the ash content was dominated by SiO2 regardless of silicate amount, and CaCO3 was a major part of the ash only when P. carterae was grown separately with the higher silicate level. These results highlight that co-culturing did not provide an advantage for improving biomass, pigments, or CaCO3 productivity.

Phosphate depletion controls lipid content and accumulation of heterotrophic bacteria during growth of Synechocystis sp. PCC 6803.

During the culturing of cyanobacteria, heterotrophic bacteria can compete for nutrients, compromise the quality of the harvested biomass, or cause culture crashes. We systematically investigated the effects of depleting inorganic phosphate (Pi) on the growth of the cyanobacterium Synechocystis sp. PCC 6803, its community of heterotrophic bacteria, and the biomass's chemical composition. On the one hand, depleting Pi had minimal impact on total biomass, extracellular polymeric substances (ESP), soluble microbial products (SMP), and most types of intracellular organic polymers production. On the other hand, depleting Pi led to markedly less lipid content, less heterotrophic biomass, and a shift in the heterotrophic community from Burkholderiales to Sphingobacteriales and Saprospirales. The causes of the large impacts were that Synechocystis was much better at scavenging a very low Pi concentration and lowering the Pi available to the heterotrophs. This work lays a foundation for controlling the accumulation of heterotrophs and reducing their deleterious effects in cyanobacteria culturing.

Electron-acceptor loadings affect chloroform dechlorination in a hydrogen-based membrane biofilm reactor.

Chloroform (CF) can undergo reductive dechlorination to dichloromethane, chloromethane, and methane. However, competition for hydrogen (H2 ), the electron-donor substrate, may cause poor dechlorination when multiple electron acceptors are present. Common acceptors in anaerobic environments are nitrate (NO3- ), sulfate (SO42- ), and bicarbonate (HCO3- ). We evaluated CF dechlorination in the presence of HCO3- at 1.56 e- Eq/m2 -day, then NO3- at 0.04-0.15 e- Eq/m2 -day, and finally NO3- (0.04 e- Eq/m2 -day) along with SO42- at 0.33 e- Eq/m2 -day in an H2 -based membrane biofilm reactor (MBfR). When the biofilm was initiated with CF-dechlorination conditions (no NO3- or SO42- ), it yielded a CF flux of 0.14 e- Eq/m2 -day and acetate production via homoacetogenesis up to 0.26 e- eq/m2 -day. Subsequent addition of NO3- at 0.05 e- Eq/m2 -day maintained full CF dechlorination and homoacetogenesis, but NO3- input at 0.15 e- Eq/m2 -day caused CF to remain in the reactor's effluent and led to negligible acetate production. The addition of SO42- did not affect CF reduction, but SO42- reduction significantly altered the microbial community by introducing sulfate-reducing Desulfovibrio and more sulfur-oxidizing Arcobacter. Dechloromonas appeared to carry out CF dechlorination and denitrification, whereas Acetobacterium (homoacetogen) may have been involved with hydrolytic dechlorination. Modifications to the electron acceptors fed to the MBfR caused the microbial community to undergo changes in structure that reflected changes in the removal fluxes.

Uptake of phosphate by Synechocystis sp. PCC 6803 in dark conditions: Removal driving force and modeling.

Rapid uptake of inorganic phosphate (Pi) by microalgae should occur through two processes operating in parallel: onto extracellular polymeric substances (EPS) and intracellular polymeric substances (IPS). Most previous studies focused only on overall Pi uptake and ignored the roles of EPS. We investigated the two-step removal of Pi by Synechocystis sp. PCC 6803 in dark conditions (i.e., without incorporation of Pi into newly synthesized biomass). We also developed a model to simulate both steps. Experimental results with Synechocystis confirmed that Pi in the bulk solution was removed by the two uptake mechanisms operating in parallel, but with different kinetics. All uptake rates decreased with time, and the Pi uptake rate by IPS was much higher than that by EPS at all times, but EPS had a larger maximum Pi-storage capacity -- 33-48 mgP/gCODEPS versus 15-17 mgP/gCODIPS. Synechocystis had a maximum Pi-storage capacity in the range of 22-28 mgP/g dry biomass. Protein in EPS and IPS played the key role for binding Pi, and biomass with higher protein content had greater Pi-storage capacity. Furthermore, biomass with low initial stored Pi had faster Pi-uptake kinetics, leading to more Pi removed from the bulk solution. This work lays the foundation for using microalgae as a means to remove Pi from polluted water and for understanding competition for Pi in microbial communities.

How myristyltrimethylammonium bromide enhances biomass harvesting and pigments extraction from Synechocystis sp. PCC 6803.

Myristyltrimethylammonium bromide (MTAB) is a cationic surfactant used to improve biomass harvesting and pigment extraction form microalgae, but the mechanisms underlying its effectiveness are poorly defined. We document the mechanisms for enhanced harvesting and pigment extraction for the cyanobacterium Synechocystis sp. PCC 6803 using measurements from flow cytometer, zeta potential, release of soluble components, and microscopy. Harvesting efficiency increased as the MTAB/Biomass dose increased from 0 to 40%. A low MTAB dose (≤ 8%) mainly brought about coagulation and flocculation, which led to aggregation that improved harvesting, but 40% MTAB had the highest harvesting efficiency, 62%. Adding MTAB above a MTAB/Biomass dose of 8% also increased cell-membrane permeability, which allowed the solvent (ethyl acetate) to pass into the cells and resulted in a large increase in extraction efficiency of pigments: An MTAB/Biomass ratio of 60% for 180 min achieved the highest extraction efficiencies of chlorophyll and carotenoids, 95% and 91%, respectively. Combining harvesting and extraction performances with results from flow cytometry, zeta potential, release of soluble components, and microscopy lead to the following mechanistic understandings. MTAB dose from 8% to 40% solubilized EPS, which lowered the biomass's negative charge, but caused breakup of the large aggregates. An increase of cell permeability also in this stage allowed ethyl acetate to pass into the cells and achieve better pigment extraction. MTAB >40% led to cell lysis and a large increase in soluble organics, but complete cell lysis was not required to achieve the maximum extraction efficiency. The MTAB/Biomass % ratio for optimizing harvest efficiency and pigment extraction lay in the range of 40%-60%.

Enhancing biodegradation of C16-alkyl quaternary ammonium compounds using an oxygen-based membrane biofilm reactor.

Quaternary ammonium compounds (QACs) (e.g., hexadecyltrimethyl-ammonium bromide, CTAB) are emerging contaminants with widespread use as surfactants and disinfectants. Because the initial step of QAC biodegradation is mono-oxygenation, QAC degraders require O2, but normal aeration leads to serious foaming. Here, we developed and tested an oxygen-based membrane biofilm reactor (O2-MBfR) that delivers O2 by diffusion through the walls of hollow-membranes to a biofilm accumulating on the outer surface of membranes. The O2-MBfR sustained QAC biodegradation even with high and toxic QAC input concentrations, up to 400 mg/L CTAB. Bubbleless O2 transfer completely eliminated foaming, and biofilm accumulation helped the QAC biodegraders resist toxicity. Pseudomonas, Achromobacter, Stenotrophomonas, and members of the Xanthomonadaceae family were dominant in the biofilm communities degrading CTAB, and their proportions depended on the O2-delivery capacity of the membranes. Bacteria capable of biodegrading QACs often harbor antibiotic resistance genes (ARGs) that help them avoid QAC toxicity. Gene copies of ARGs were detected in biofilms and liquid, but the levels of ARGs were 5- to 35-fold lower in the liquid than in the biofilm. In summary, the O2-MBfR achieved aerobic biodegradation of CTAB with neither foaming nor toxicity, and it also minimized the spread of ARGs.

The distribution of phosphorus and its transformations during batch growth of Synechocystis.

Phosphorus (P) is an essential nutrient that affects the growth and metabolism of microalgal biomass. Despite the obvious importance of P, the dynamics of how it is taken up and distributed in microalgae are largely undefined. In this study, we tracked the fate of P during batch growth of the cyanobacterium Synechocystis sp. PCC 6803. We determined the distribution of P in intracellular polymeric substances (IPS), extracellular polymeric substances (EPS), and soluble microbial products (SMP) for three initial ortho-phosphate concentrations. Results show that the initial P concentration had no impact on the production of biomass, SMP, and EPS. While the initial P concentration affected the rate and the timing of how P was transformed among internal and external forms of inorganic P (IP) and organic P (OP), the trends were the same no matter the starting P concentration. Initially, IP in the bulk solution was rapidly and simultaneously adsorbed by EPS (IPEPS) and taken up as internal IP (IPint). As the bulk-solution's IP was depleted, desorption of IPEPS became the predominant source for IP that was taken up by the growing cells and converted into OPint. At the end of the 9-d batch experiments, almost all P was OP, and most of the OP was intracellular. Based on all of the results, we propose a set of transformation pathways for P during the growth of Synechocystis. Key is that EPS and intracellular P pool play important and distinct roles in the uptake and storage of P.

Selective fermentation of carbohydrate and protein fractions of Scenedesmus, and biohydrogenation of its lipid fraction for enhanced recovery of saturated fatty acids.

Biofuels derived from microalgae have promise as carbon-neutral replacements for petroleum. However, difficulty extracting microalgae-derived lipids and the co-extraction of non-lipid components add major costs that detract from the benefits of microalgae-based biofuel. Selective fermentation could alleviate these problems by managing microbial degradation so that carbohydrates and proteins are hydrolyzed and fermented, but lipids remain intact. We evaluated selective fermentation of Scenedesmus biomass in batch experiments buffered at pH 5.5, 7, or 9. Carbohydrates were fermented up to 45% within the first 6 days, protein fermentation followed after about 20 days, and lipids (measured as fatty acid methyl esters, FAME) were conserved. Fermentation of the non-lipid components generated volatile fatty acids, with acetate, butyrate, and propionate being the dominant products. Selective fermentation of Scenedesmus biomass increased the amount of extractable FAME and the ratio of FAME to crude lipids. It also led to biohydrogenation of unsaturated FAME to more desirable saturated FAME (especially to C16:0 and C18:0), and the degree of saturation was inversely related to the accumulation of hydrogen gas after fermentation. Moreover, the microbial communities after selective fermentation were enriched in bacteria from families known to perform biohydrogenation, i.e., Porphyromonadaceae and Ruminococcaceae. Thus, this study provides proof-of-concept that selective fermentation can improve the quantity and quality of lipids that can be extracted from Scenedesmus.

Effects of pulsed electric field treatment on enhancing lipid recovery from the microalga, Scenedesmus.

Chloroform and methanol are superior solvents for lipid extraction from photosynthetic microorganisms, because they can overcome the resistance offered by the cell walls and membranes, but they are too toxic and expensive to use for large-scale fuel production. Biomass from the photosynthetic microalga Scenedesmus, subjected to a commercially available pre-treatment technology called Focused-Pulsed® (FP), yielded 3.1-fold more crude lipid and fatty acid methyl ester (FAME) after extraction with a range of solvents. FP treatment increased the FAME-to-crude-lipid ratio for all solvents, which means that the extraction of non-lipid materials was minimized, while the FAME profile itself was unchanged compared to the control. FP treatment also made it possible to use only a small proportion of chloroform and methanol, along with isopropanol, to obtain equivalent yields of lipid and FAME as with 100% chloroform plus methanol.