Robin Gerlach

In silico approaches to study mass and energy flows in microbial consortia: a syntrophic case study

BackgroundThree methods were developed for the application of stoichiometry-based network analysis approaches including elementary mode analysis to the study of mass and energy flows in microbial communities. Each has distinct advantages and disadvantages suitable for analyzing systems with different degrees of complexity and a priori knowledge. These approaches were tested and compared using data from the thermophilic, phototrophic mat communities from Octopus and Mushroom Springs in Yellowstone National Park (USA). The models were based on three distinct microbial guilds: oxygenic phototrophs, filamentous anoxygenic phototrophs, and sulfate-reducing bacteria. Two phases, day and night, were modeled to account for differences in the sources of mass and energy and the routes available for their exchange.ResultsThe in silico models were used to explore fundamental questions in ecology including the prediction of and explanation for measured relative abundances of primary producers in the mat, theoretical tradeoffs between overall productivity and the generation of toxic by-products, and the relative robustness of various guild interactions.ConclusionThe three modeling approaches represent a flexible toolbox for creating cellular metabolic networks to study microbial communities on scales ranging from cells to ecosystems. A comparison of the three methods highlights considerations for selecting the one most appropriate for a given microbial system. For instance, communities represented only by metagenomic data can be modeled using the pooled method which analyzes a communitys total metabolic potential without attempting to partition enzymes to different organisms. Systems with extensive a priori information on microbial guilds can be represented using the compartmentalized technique, employing distinct control volumes to separate guild-appropriate enzymes and metabolites. If the complexity of a compartmentalized network creates an unacceptable computational burden, the nested analysis approach permits greater scalability at the cost of more user intervention through multiple rounds of pathway analysis.

An efficient and scalable extraction and quantification method for algal derived biofuel.

Microalgae are capable of synthesizing a multitude of compounds including biofuel precursors and other high value products such as omega-3-fatty acids. However, accurate analysis of the specific compounds produced by microalgae is important since slight variations in saturation and carbon chain length can affect the quality, and thus the value, of the end product. We present a method that allows for fast and reliable extraction of lipids and similar compounds from a range of algae, followed by their characterization using gas chromatographic analysis with a focus on biodiesel-relevant compounds. This method determines which range of biologically synthesized compounds is likely responsible for each fatty acid methyl ester (FAME) produced; information that is fundamental for identifying preferred microalgae candidates as a biodiesel source. Traditional methods of analyzing these precursor molecules are time intensive and prone to high degrees of variation between species and experimental conditions. Here we detail a new method which uses microwave energy as a reliable, single-step cell disruption technique to extract lipids from live cultures of microalgae. After extractable lipid characterization (including lipid type (free fatty acids, mono-, di- or tri-acylglycerides) and carbon chain length determination) by GC-FID, the same lipid extracts are transesterified into FAMEs and directly compared to total biodiesel potential by GC-MS. This approach provides insight into the fraction of total FAMEs derived from extractable lipids compared to FAMEs derived from the residual fraction (i.e. membrane bound phospholipids, sterols, etc.). This approach can also indicate which extractable lipid compound, based on chain length and relative abundance, is responsible for each FAME. This method was tested on three species of microalgae; the marine diatom Phaeodactylum tricornutum, the model Chlorophyte Chlamydomonas reinhardtii, and the freshwater green alga Chlorella vulgaris. The method is shown to be robust, highly reproducible, and fast, allowing for multiple samples to be analyzed throughout the time course of culturing, thus providing time-resolved information regarding lipid quantity and quality. Total time from harvesting to obtaining analytical results is less than 2h.

Direct measurement and characterization of active photosynthesis zones inside wastewater remediating and biofuel producing microalgal biofilms.

Microalgal biofilm based technologies are of keen interest due to their high biomass concentrations and ability to utilize light and CO2. While photoautotrophic biofilms have long been used for wastewater remediation, biofuel production represents a relatively new and under-represented focus area. However, the direct measurement and characterization of fundamental parameters required for industrial control are challenging due to biofilm heterogeneity. This study evaluated oxygenic photosynthesis and respiration on two distinct microalgal biofilms cultured using a novel rotating algal biofilm reactor operated at field- and laboratory-scales. Clear differences in oxygenic photosynthesis and respiration were observed based on different culturing conditions, microalgal composition, light intensity and nitrogen availability. The cultures were also evaluated as potential biofuel synthesis strategies. Nitrogen depletion was not found to have the same effect on lipid accumulation compared to traditional planktonic microalgal studies. Physiological characterizations of these microalgal biofilms identify fundamental parameters needed to understand and control process optimization.

Fracture Sealing with Microbially-Induced Calcium Carbonate Precipitation: A Field Study

A primary environmental risk from unconventional oil and gas development or carbon sequestration is subsurface fluid leakage in the near wellbore environment. A potential solution to remediate leakage pathways is to promote microbially induced calcium carbonate precipitation (MICP) to plug fractures and reduce permeability in porous materials. The advantage of microbially induced calcium carbonate precipitation (MICP) over cement-based sealants is that the solutions used to promote MICP are aqueous. MICP solutions have low viscosities compared to cement, facilitating fluid transport into the formation. In this study, MICP was promoted in a fractured sandstone layer within the Fayette Sandstone Formation 340.8 m below ground surface using conventional oil field subsurface fluid delivery technologies (packer and bailer). After 24 urea/calcium solution and 6 microbial (Sporosarcina pasteurii) suspension injections, the injectivity was decreased (flow rate decreased from 1.9 to 0.47 L/min) and a reduction in the in-well pressure falloff (>30% before and 7% after treatment) was observed. In addition, during refracturing an increase in the fracture extension pressure was measured as compared to before MICP treatment. This study suggests MICP is a promising tool for sealing subsurface fractures in the near wellbore environment.

Optimized inorganic carbon regime for enhanced growth and lipid accumulation in Chlorella vulgaris

BackgroundLarge-scale algal biofuel production has been limited, among other factors, by the availability of inorganic carbon in the culture medium at concentrations higher than achievable with atmospheric CO2. Life cycle analyses have concluded that costs associated with supplying CO2 to algal cultures are significant contributors to the overall energy consumption.ResultsA two-phase optimal growth and lipid accumulation scenario is presented, which (1) enhances the growth rate and (2) the triacylglyceride (TAG) accumulation rate in the oleaginous Chlorophyte Chlorella vulgaris strain UTEX 395, by growing the organism in the presence of low concentrations of NaHCO3 (5 mM) and controlling the pH of the system with a periodic gas sparge of 5 % CO2 (v/v). Once cultures reached the desired cell densities, which can be “fine-tuned” based on initial nutrient concentrations, cultures were switched to a lipid accumulation metabolism through the addition of 50 mM NaHCO3. This two-phase approach increased the specific growth rate of C. vulgaris by 69 % compared to cultures sparged continuously with 5 % CO2 (v/v); further, biomass productivity (g L−1 day−1) was increased by 27 %. Total biodiesel potential [assessed as total fatty acid methyl ester (FAME) produced] was increased from 53.3 to 61 % (FAME biomass−1) under the optimized conditions; biodiesel productivity (g FAME L−1 day−1) was increased by 7.7 %. A bicarbonate salt screen revealed that American Chemical Society (ACS) and industrial grade NaHCO3 induced the highest TAG accumulation (% w/w), whereas Na2CO3 did not induce significant TAG accumulation. NH4HCO3 had a negative effect on cell health presumably due to ammonia toxicity. The raw, unrefined form of trona, NaHCO3∙Na2CO3 (sodium sesquicarbonate) induced TAG accumulation, albeit to a slightly lower extent than the more refined forms of sodium bicarbonate.ConclusionsThe strategic addition of sodium bicarbonate was found to enhance growth and lipid accumulation rates in cultures of C. vulgaris, when compared to traditional culturing strategies, which rely on continuously sparging algal cultures with elevated concentrations of CO2(g). This work presents a two-phased, improved photoautotrophic growth and lipid accumulation approach, which may result in an overall increase in algal biofuel productivity.

Dissolved inorganic carbon enhanced growth, nutrient uptake, and lipid accumulation in wastewater grown microalgal biofilms

Microalgal biofilms grown to evaluate potential nutrient removal options for wastewaters and feedstock for biofuels production were studied to determine the influence of bicarbonate amendment on their growth, nutrient uptake capacity, and lipid accumulation after nitrogen starvation. No significant differences in growth rates, nutrient removal, or lipid accumulation were observed in the algal biofilms with or without bicarbonate amendment. The biofilms possibly did not experience carbon-limited conditions because of the large reservoir of dissolved inorganic carbon in the medium. However, an increase in photosynthetic rates was observed in algal biofilms amended with bicarbonate. The influence of bicarbonate on photosynthetic and respiration rates was especially noticeable in biofilms that experienced nitrogen stress. Medium nitrogen depletion was not a suitable stimulant for lipid production in the algal biofilms and as such, focus should be directed toward optimizing growth and biomass productivities to compensate for the low lipid yields and increase nutrient uptake.

Construction of two ureolytic model organisms for the study of microbially induced calcium carbonate precipitation

Two bacterial strains, Pseudomonas aeruginosa MJK1 and Escherichia coli MJK2, were constructed that both express green fluorescent protein (GFP) and carry out ureolysis. These two novel model organisms are useful for studying bacterial carbonate mineral precipitation processes and specifically ureolysis-driven microbially induced calcium carbonate precipitation (MICP). The strains were constructed by adding plasmid-borne urease genes (ureABC, ureD and ureFG) to the strains P. aeruginosa AH298 and E. coli AF504gfp, both of which already carried unstable GFP derivatives. The ureolytic activities of the two new strains were compared to the common, non-GFP expressing, model organism Sporosarcina pasteurii in planktonic culture under standard laboratory growth conditions. It was found that the engineered strains exhibited a lower ureolysis rate per cell but were able to grow faster and to a higher population density under the conditions of this study. Both engineered strains were successfully grown as biofilms in capillary flow cell reactors and ureolysis-induced calcium carbonate mineral precipitation was observed microscopically. The undisturbed spatiotemporal distribution of biomass and calcium carbonate minerals were successfully resolved in 3D using confocal laser scanning microscopy. Observations of this nature were not possible previously because no obligate urease producer that expresses GFP had been available. Future observations using these organisms will allow researchers to further improve engineered application of MICP as well as study natural mineralization processes in model systems.

Using life cycle assessment and techno-economic analysis in a real options framework to inform the design of algal biofuel production facilities

This study investigates the use of real options analysis (ROA) to quantify the value of greater product flexibility at algal biofuel production facilities. A deterministic optimization framework is integrated with a combined life cycle assessment/techno-economic analysis model and subjected to an ensemble of 30-year commodity price trajectories. Profits are maximized for two competing plant configurations: 1) one that sells lipid-extracted algae as animal feed only; and 2) one that can sell lipid-extracted algae as feed or use it to recover nutrients and energy, due to an up-front investment in anaerobic digestion/combined heat and power. Results show that added investment in plant flexibility does not result in an improvement in net present value, because current feed meal prices discourage use of lipid-extracted algae for nutrient and energy recovery. However, this study demonstrates that ROA provides many useful insights regarding plant design that cannot be captured via traditional techno-economic modeling.

Effect of selenite on the morphology and respiratory activity of Phanerochaete chrysosporium biofilms

The temporal and spatial effects of selenite (SeO3(2-)) on the physical properties and respiratory activity of Phanerochaete chrysosporium biofilms, grown in flow-cell reactors, were investigated using oxygen microsensors and confocal laser scanning microscopy (CLSM) imaging. Exposure of the biofilm to a SeO3(2-) load of 1.67mgSeL(-1)h(-1) (10mgSeL(-1) influent concentration), for 24h, resulted in a 20% reduction of the O2 flux, followed by a ∼10% decrease in the glucose consumption rate. Long-term exposure (4days) to SeO3(2-) influenced the architecture of the biofilm by creating a more compact and dense hyphal arrangement resulting in a decrease of biofilm thickness compared to fungal biofilms grown without SeO3(2-). To the best of our knowledge, this is the first time that the effect of SeO3(2-) on the aerobic respiratory activity on fungal biofilms is described.

Evaluating the relative impacts of operational and financial factors on the competitiveness of an algal biofuel production facility

Algal biofuels are becoming more economically competitive due to technological advances and government subsidies offering tax benefits and lower cost financing. These factors are linked, however, as the value of technical advances is affected by modeling assumptions regarding the growth conditions, process design, and financing of the production facility into which novel techniques are incorporated. Two such techniques, related to algal growth and dewatering, are evaluated in representative operating and financing scenarios using an integrated techno-economic model. Results suggest that these techniques can be valuable under specified conditions, but also that investment subsidies influence cost competitive facility design by incentivizing development of more capital intensive facilities (e.g., favoring hydrothermal liquefaction over transesterification-based facilities). Evaluating novel techniques under a variety of operational and financial scenarios highlights the set of site-specific conditions in which technical advances are most valuable, while also demonstrating the influence of subsidies linked to capital intensity.