Phase partitioning of mercury, arsenic, selenium, and cadmium in Chlamydomonas reinhardtii and Arthrospira maxima microcosms.

As the global human population increases, demand for protein will surpass our current production ability without an increase in land use or intensification. Microalgae cultivation offers a high yield of protein, and utilization of wastewater from municipal or agricultural sources in place of freshwater for microalgae aquaculture may increase the sustainability of this practice. However, wastewater from municipal and agricultural sources may contain contaminants, such as mercury (Hg), cadmium (Cd), selenium (Se), and arsenic (As). Association of these elements with algal biomass may present an exposure risk to product consumers, while volatilization may present an exposure hazard to industry workers. Thus, the partitioning of these elements should be evaluated before wastewater can be confidently used in an aquaculture setting. This study explored the potential for exposure associated with Arthrospira maxima and Chlamydomonas reinhardtii aquaculture in medium contaminated with 0.33 µg Hg L-1, 60 µg As L-1, 554 µg Se L-1, and 30 µg Cd L-1. Gaseous effluent from microalgae aquaculture was analyzed for Hg, As, Se, and Cd to quantify volatilization. A mass balance approach was used to describe the partitioning of elements between the biomass, medium, and gas phases at the end of exponential growth. Contaminants were recovered predominantly in medium and biomass, regardless of microalgae strain. In the case of Hg, 48 ± 2% was associated with A. maxima biomass and 55 ± 8% with C. reinhardtii when Hg was present as the only contaminant, but this increased to 85 ± 11% in C. reinhardtii biomass when As, Se, and Cd were also present. A small and highly variable abiotic volatilization of Hg was observed in the gas phase of both A. maxima and C. reinhardtii cultures. Evidence presented herein suggests that utilizing wastewater containing Hg, Cd, Se, and As for microalgae cultivation may present health hazards to consumers.

Assessing the environmental impact of resource recovery from dairy manure.

Manure management is a major contributor to environmental impacts from large-scale dairy production. In this study, technologies for recovering energy, nutrients, and water from dairy manure were evaluated using life cycle assessment (LCA) and compared to conventional practices on California dairy farms. Six scenarios were evaluated: conventional manure management practices, anaerobic digestion (AD) for biogas recovery, and four scenarios for nutrients, energy, and water integrated recovery, called NEWIR. The NEWIR system consists of hydrothermal carbonization (HTC) for energy recovery via hydrochar, algae cultivation in the HTC aqueous product for nutrient recovery and production of protein-rich cattle feed, and water recovery from algae pond effluent via membrane distillation. Four NEWIR scenarios were evaluated, each with a different species of algae. Based on the results of the LCA, AD improves GHG emissions relative to conventional practices by 82%, but has similar eutrophication impacts, posing similar concerns for nutrient management as current practices. Results for the NEWIR system are highly dependent on the algae species used. Three of the four species evaluated (Chlamydomonas reinhardtii, Chlorella vulgaris, and Scenedesmus obliquus) improve GHG emissions by 420-500 kg CO2-eq. per functional unit, while net water consumption is increased by approximately 75% over AD and conventional practices Spirulina maxima requires more water and chemical inputs for cultivation than the other species, resulting in higher water use (21 times higher than baseline), though GHG emissions are still reduced by 85 kg CO2-eq. per functional unit relative to conventional practices. All NEWIR scenarios improve eutrophication impacts relative to AD and conventional practices by 16-46% for marine eutrophication and 18-99% for freshwater eutrophication, depending on the algae species used. The results suggest integrated resource recovery through NEWIR is a promising treatment method for manure to mitigate GHG emissions and improve nutrient management on large-scale farms. In addition, carbon and nutrient trading policies are discussed in relation to resource recovery technologies and their potential to incentivize producers to recover products from dairy manure.

Nutrient recovery of the hydrothermal carbonization aqueous product from dairy manure using membrane distillation.

Phosphorus is a crucial resource for the agricultural industry, but its limited supply requires recovery from waste materials before it is lost and leads to eutrophication. Dairy manure is rich with phosphorus, and the growth and consolidation within the dairy industry has led to dairy manure management becoming a significant concern. Hydrothermal carbonization (HTC) and membrane distillation (MD) were investigated as an alternative to treat dairy manure and recover nutrients, specifically phosphorus and nitrogen. HTC is a thermal treatment process that converts organic matter into a hydrochar analogous to a low-grade coal, and MD is a thermally-driven separation process that can utilize low-grade waste heat from HTC, thus the two processes are synergetic. A byproduct of the HTC process is the aqueous product (HAP) that contains the water-soluble nutrients and organic components of dairy manure. In this work, the efficacy of MD to concentrate the nutrients in the presence of dissolved organic carbon was assessed. Samples included synthetic nutrient-rich streams as well as HAP produced at HTC temperatures ranging from 200 °C to 260 °C. In each case, the nutrients were successfully concentrated in the feed loop with rejections >99%. Dissolved carbon was found to foul the MD membrane at levels proportional to its hydrophobicity, with little fouling observed for glucose and substantial fouling observed for HAP solutions created at higher temperatures.

High frequency dielectrophoretic response of microalgae over time.

The high frequency dielectrophoresis (>20 MHz) response of microalgae cells with different lipid content was monitored over time. Chlamydomonas reinhardtii was cultured in regular medium and under nitrogen-depleted conditions in order to produce populations of cells with low and high lipid content, respectively. The electrical conductivity of the culture media was also monitored over the same time. The upper crossover frequency decreased for high-lipid cells over time. The single-shell model predicts that the upper crossover frequency is dictated primarily by the dielectric properties of the cytoplasm. The high frequency DEP response of the high-lipid cells' cytoplasm was changed by lipid accumulation. DEP response of the low-lipid cells also varied with the conductivity of the culture media due to nutrient consumption. Relative lipid content was estimated with BODIPY 505/515 dye by calculating the area-weighted intensity average of fluorescent images. Finally, microalgae cells were successfully separated based on lipid content at 41 MHz and DEP media conductivity 106 ± 1 µS/cm.

Molecular assessment of the sensitivity of sulfate-reducing microbial communities remediating mine drainage to aerobic stress.

Sulfate-reducing permeable reactive zones (SR-PRZs) are microbially-driven anaerobic systems designed for the removal of heavy metals and sulfate in mine drainage. Environmental perturbations, such as oxygen exposure, may adversely affect system stability and long-term performance. The objective of this study was to examine the effect of two successive aerobic stress events on the performance and microbial community composition of duplicate laboratory-scale lignocellulosic SR-PRZs operated using the following microbial community management strategies: biostimulation with ethanol or carboxymethylcellulose; bioaugmentation with sulfate-reducing or cellulose-degrading enrichments; inoculation with dairy manure only; and no inoculation. A functional gene-based approach employing terminal restriction fragment length polymorphism and quantitative polymerase chain reaction targeting genes of sulfate-reducing (dsrA), cellulose-degrading (cel5, cel48), fermentative (hydA), and methanogenic (mcrA) microbes was applied. In terms of performance (i.e., sulfate removal), biostimulation with ethanol was the only strategy that clearly had an effect (positive) following exposure to oxygen. In terms of microbial community composition, significant shifts were observed over the course of the experiment. Results suggest that exposure to oxygen more strongly influenced microbial community shifts than the different microbial community management strategies. Sensitivity to oxygen exposure varied among different populations and was particularly pronounced for fermentative bacteria. Although the community structure remained altered after exposure, system performance recovered, indicating that SR-PRZ microbial communities were functionally redundant. Results suggest that pre-exposure to oxygen might be a more effective strategy to improve the resilience of SR-PRZ microbial communities relative to bioaugmentation or biostimulation.

Effect of bioaugmentation and biostimulation on sulfate-reducing column startup captured by functional gene profiling.

Sulfate-reducing permeable reactive zones (SR-PRZs) depend upon a complex microbial community to utilize a lignocellulosic substrate and produce sulfides, which remediate mine drainage by binding heavy metals. To gain insight into the impact of the microbial community composition on the startup time and pseudo-steady-state performance, functional genes corresponding to cellulose-degrading (CD), fermentative, sulfate-reducing, and methanogenic microorganisms were characterized in columns simulating SR-PRZs using quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE). Duplicate columns were bioaugmented with sulfate-reducing or CD bacteria or biostimulated with ethanol or carboxymethyl cellulose and compared with baseline dairy manure inoculum and uninoculated controls. Sulfate removal began after ~ 15 days for all columns and pseudo-steady state was achieved by Day 30. Despite similar performance, DGGE profiles of 16S rRNA gene and functional genes at pseudo-steady state were distinct among the column treatments, suggesting the potential to control ultimate microbial community composition via bioaugmentation and biostimulation. qPCR revealed enrichment of functional genes in all columns between the initial and pseudo-steady-state time points. This is the first functional gene-based study of CD, fermentative and sulfate-reducing bacteria and methanogenic archaea in a lignocellulose-based environment and provides new qualitative and quantitative insight into startup of a complex microbial system.

Active community profiling via capillary electrophoresis single-strand conformation polymorphism analysis of amplified 16S rRNA and 16S rRNA genes.

Here, we report the validation and advancement of a high-throughput method for fingerprinting the active members of a microbial community. This method, termed active community profiling (ACP), provides information about both the composition and the activity of mixed microbial cultures via comparative measurements of amplified 16S rRNA (RNA) and 16S rRNA genes (DNA). Capillary electrophoresis is used to resolve single-strand conformation polymorphisms of polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) products, producing electropherograms representative of the community structure. Active members of the community are distinguished by elevated RNA:DNA peak area ratios. Chemostat experiments with defined populations were conducted to validate the ACP approach. Using a pure culture of Escherichia coli, a direct correlation was found between the growth rate and the RNA:DNA peak ratio. In a second validation experiment, a binary culture of E. coli and Pseudomonas putida was subjected to a controlled environmental change consisting of a shift to anaerobic conditions. ACP revealed the expected cessation of growth of P. putida, an obligate aerobe, while the corresponding DNA-only analysis indicated no change in the culture. Finally, ACP was applied to a complex microbial community, and a novel binning approach was demonstrated for integrating the RNA and DNA electropherograms. ACP thus represents a significant advance from traditional DNA-based profiling techniques, which do not distinguish active from inactive or dead cells, and is well suited for high-throughput community analysis.

Microbial community analysis of two field-scale sulfate-reducing bioreactors treating mine drainage.

The microbial communities of two field-scale pilot sulfate-reducing bioreactors treating acid mine drainage (AMD), Luttrell and Peerless Jenny King (PJK), were compared using biomolecular tools and multivariate statistical analyses. The two bioreactors were well suited for this study because their geographic locations and substrate compositions were similar while the characteristics of influent AMD, configuration and degree of exposure to oxygen were distinct. The two bioreactor communities were found to be functionally similar, including cellulose degraders, fermenters and sulfate-reducing bacteria (SRB). Significant differences were found between the two bioreactors in phylogenetic comparisons of cloned 16S rRNA genes and adenosine 5'-phosphosulfate reductase (apsA) genes. The apsA gene clones from the Luttrell bioreactor were dominated by uncultured SRB most closely related to Desulfovibrio spp., while those of the PJK bioreactor were dominated by Thiobacillus spp. The fraction of the SRB genus Desulfovibrio was also higher at Luttrell than at PJK as determined by quantitative real-time polymerase chain reaction analysis. Oxygen exposure at PJK is hypothesized to be the primary cause of these differences. This study is the first rigorous phylogenetic investigation of field-scale bioreactors treating AMD and the first reported application of multivariate statistical analysis of remediation system microbial communities applying UniFrac software.