Brendan T. Higgins

MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis

Data-independent acquisition (DIA) in liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) provides comprehensive untargeted acquisition of molecular data. We provide an open-source software pipeline, which we call MS-DIAL, for DIA-based identification and quantification of small molecules by mass spectral deconvolution. For a reversed-phase LC-MS/MS analysis of nine algal strains, MS-DIAL using an enriched LipidBlast library identified 1,023 lipid compounds, highlighting the chemotaxonomic relationships between the algal strains.

Effects of Escherichia coli on Mixotrophic Growth of Chlorella minutissima and Production of Biofuel Precursors

Chlorella minutissima was co-cultured with Escherichia coli in airlift reactors under mixotrophic conditions (glucose, glycerol, and acetate substrates) to determine possible effects of bacterial contamination on algal biofuel production. It was hypothesized that E. coli would compete with C. minutissima for nutrients, displacing algal biomass. However, C. minutissima grew more rapidly and to higher densities in the presence of E. coli, suggesting a symbiotic relationship between the organisms. At an initial 1% substrate concentration, the co-culture produced 200-587% more algal biomass than the axenic C. minutissima cultures. Co-cultures grown on 1% substrate consumed 23–737% more of the available carbon substrate than the sum of substrate consumed by E. coli and C. minutissima alone. At 1% substrate, total lipid and starch productivity were elevated in co-cultures compared to axenic cultures indicating that bacterial contamination was not detrimental to the production of biofuel precursors in this specific case. Bio-fouling of the reactors observed in co-cultures and acid formation in all mixotrophic cultures, however, could present challenges for scale-up.

Life Cycle Environmental and Cost Impacts of Using an Algal Turf Scrubber to Treat Dairy Wastewater

Using algae to simultaneously treat wastewater and produce energy products has potential environmental and economic benefits. This study evaluates the life cycle energy, greenhouse gas (GHG) emissions, eutrophication potential, and cost impacts of incorporating an algal turf scrubber (ATS) into a treatment process for dairy wastewater. A life cycle inventory and cost model was developed to simulate an ATS treatment system where harvested algae would be used to generate biogas for process heat and electricity generation. Modeling results show that using an ATS significantly reduces eutrophication impacts by reducing chemical oxygen demand, nitrogen, and phosphorus in the wastewater. With low water recirculation rates through the ATS and high algae productivity, inclusion of the ATS results in net energy displacement and a reduction of GHG emissions compared to a system with no ATS. However, if high water recirculation rates are used or if algae biosolids from the digester are dried, the system results in a net increase in energy consumption and GHG emissions. The life cycle treatment cost was estimated to be

Microplate assay for quantitation of neutral lipids in extracts from microalgae

1.42 USD per cubic meter of treated wastewater. At this cost, using an ATS would only be cost effective for dairies if they received monetary credits for improved water quality on the order of

Co-culturing Chlorella minutissima with Escherichia coli can increase neutral lipid production and improve biodiesel quality.

3.83 per kilogram of nitrogen and

Algal–bacterial synergy in treatment of winery wastewater

9.57 per kilogram of phosphorus through, for example, nutrient trading programs.

Ionic Liquids Impact the Bioenergy Feedstock-Degrading Microbiome and Transcription of Enzymes Relevant to Polysaccharide Hydrolysis

Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC-MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 μg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.

Cofactor symbiosis for enhanced algal growth, biofuel production, and wastewater treatment

Lipid productivity and fatty acid composition are important metrics for the production of high quality biodiesel from algae. Our previous results showed that co‐culturing the green alga Chlorella minutissima with Escherichia coli under high‐substrate mixotrophic conditions enhanced both culture growth and crude lipid content. To investigate further, we analyzed neutral lipid content and fatty acid content and composition of axenic cultures and co‐cultures produced under autotrophic and mixotrophic conditions. We found that co‐culturing C. minutissima with E. coli under high substrate conditions (10 g/L) increased neutral lipid content 1.9‐ to 3.1‐fold and fatty acid content 1.5‐ to 2.6‐fold compared to equivalent axenic C. minutissima cultures. These same co‐cultures also exhibited a significant fatty acid shift away from trienoic and toward monoenoic fatty acids thereby improving the quality of the synthesized fatty acids for biodiesel production. Further investigation suggested that E. coli facilitates substrate uptake by the algae and that the resulting growth enhancement induces a nitrogen‐limited condition. Enhanced carbon uptake coupled with nitrogen limitation is the likely cause of the observed neutral lipid accumulation and fatty acid profile changes. Biotechnol. Bioeng. 2015;112: 1801–1809.

The role of organic matter amendment level on soil heating, organic acid accumulation, and development of bacterial communities in solarized soil

There is significant potential for employing algae in tertiary wastewater treatment, however, little is known about the contribution of algae-bacteria synergy toward treatment performance. This study demonstrates potential synergy in the treatment of three winery wastewater samples. Two strains of green algae, Auxenochlorella protothecoides and Chlorella sorokiniana were tested and each removed > 90% of nitrogen, > 50% of phosphate, and 100% of acetic acid in the wastewater. Both algae strains grew significantly faster on wastewaters compared to growth on minimal media. Organic carbon in the wastewater apparently played a limited role in algal growth enhancement. When cultured on sterile-filtered wastewater, A. protothecoides increased soluble COD loadings in two of the three wastewaters and C. sorokiniana secreted an insoluble film. Culturing algae with the native wastewater microbial community negated the secretion of algal photosynthate, allowing for simultaneous reductions in COD and nutrient concentrations. Both algae species stimulated bacterial growth in a strain-specific way, suggesting unique responses to algal photosynthate. Cofactor auxotrophy for thiamine, cobalamin, and biotin is widespread among algae and these cofactors are typically obtained from bacteria. Sequencing the wastewater microbial community revealed bacteria capable of synthesizing all three cofactors while liquid chromatography with mass spectrometry (LCMS) and bio-assays revealed the presence of thiamine metabolites in the wastewaters. These cofactors likely increased algal growth rates, particularly for A. protothecoides, which cannot synthesize thiamine de-novo but can salvage it from degradation products. Collectively, these results demonstrate that bacteria and algae provided synergistic growth benefits, potentially contributing to higher levels of wastewater treatment than either organism type alone.Winery wastewater: Working in tandemBacteria and algae work in synergy to treat wastewater from wineries. Anaerobic digesters are used in the food processing industry to reduce organic content in wastewater, but are ineffective at removing inorganic nutrients. Algae, on the other hand, digest ammonium and phosphates, but secrete undesirable organic materials. A team led by Brendan Higgins at Auburn University in the US look at how bacteria and algae respond to one another when both are used to treat winery wastewater. Encouragingly, they find that the algae effectively remove nitrogen, phosphates, sulfates and acetic acid while simultaneously enhancing bacterial growth, and the bacteria in turn reduces the organic content, including the algae-produced photosynthate. Synergies between algae and bacteria thus show great potential for wastewater treatment.

Informatics for improved algal taxonomic classification and research: A case study of UTEX 2341

Pretreatment using ionic liquids (IL) is a promising approach for the conversion of lignocellulose to biofuels. Because IL can be inhibitory to enzymes and microorganisms involved in downstream hydrolysis and fermentation steps, discovery of IL-tolerant organisms and enzymes is critical for advancing this technology. Employing metatranscriptomics in the analysis of IL-enriched cultures facilitated tracking of dynamic changes in a complex microbial community at the level of gene transcription and doing so with genome resolution. Specific organisms were discovered that could simultaneously tolerate a moderate IL concentration and transcribe a diverse array of cellulolytic enzymes. Gene sequences of cellulolytic enzymes and efflux pumps from those same organisms were also identified, providing important resources for future research on engineering IL-tolerant organisms and enzymes. ABSTRACT Ionic liquid (IL) pretreatment is a promising approach for the conversion of lignocellulose to biofuels. The toxicity of residual IL, however, negatively impacts the performance of industrial enzymes and microorganisms in hydrolysis and fermentation. In this study, a thermophilic microbial community was cultured on switchgrass amended with various levels of the ionic liquid 1-ethyl-3-methylimidazolium acetate. Changes in the microbial community composition and transcription of genes relevant to IL tolerance and lignocellulose hydrolysis were quantified. Increasing the level of IL to 0.1% (wt) led to increased levels of relative abundance and transcription in organisms of the phylum Firmicutes. Interestingly, IL concentrations of up to 1% (wt) also resulted in greater xylanase transcription and enzyme activity as well as increased transcription of endoglucanase, beta-glucosidase, and IL tolerance genes compared to communities without IL. IL levels above 1% (wt) resulted in decreased enzyme activity and transcription of genes involved in lignocellulose hydrolysis. The results indicate that moderate levels of IL select for thermophilic microorganisms that not only tolerate IL but also effectively hydrolyze lignocellulose from switchgrass. Discovery of IL-tolerant organisms and enzymes is critical for the development of biological processes that convert IL-pretreated biomass to biofuels and chemicals. Employing metatranscriptomic analysis of enrichment cultures can facilitate the discovery of microorganisms and enzymes that may be active in the presence of toxic compounds such as ionic liquids. IMPORTANCE Pretreatment using ionic liquids (IL) is a promising approach for the conversion of lignocellulose to biofuels. Because IL can be inhibitory to enzymes and microorganisms involved in downstream hydrolysis and fermentation steps, discovery of IL-tolerant organisms and enzymes is critical for advancing this technology. Employing metatranscriptomics in the analysis of IL-enriched cultures facilitated tracking of dynamic changes in a complex microbial community at the level of gene transcription and doing so with genome resolution. Specific organisms were discovered that could simultaneously tolerate a moderate IL concentration and transcribe a diverse array of cellulolytic enzymes. Gene sequences of cellulolytic enzymes and efflux pumps from those same organisms were also identified, providing important resources for future research on engineering IL-tolerant organisms and enzymes.