Stefanie Van Wychen

Algal Biomass Constituent Analysis: Method Uncertainties and Investigation of the Underlying Measuring Chemistries

Algal biomass compositional analysis data form the basis of a large number of techno-economic process analysis models that are used to investigate and compare different processes in algal biofuels production. However, the analytical methods used to generate these data are far from standardized. This work investigated the applicability of common methods for rapid chemical analysis of biomass samples with respect to accuracy and precision. This study measured lipids, protein, carbohydrates, ash, and moisture of a single algal biomass sample at 3 institutions by 8 independent researchers over 12 separate workdays. Results show statistically significant differences in the results from a given analytical method among laboratories but not between analysts at individual laboratories, suggesting consistent training is a critical issue for empirical analytical methods. Significantly different results from multiple lipid and protein measurements were found to be due to different measurement chemistries. We identified a set of compositional analysis procedures that are in best agreement with data obtained by more advanced analytical procedures. The methods described here and used for the round robin experiment do not require specialized instrumentation, and with detailed analytical documentation, the differences between laboratories can be markedly reduced.

Separation and quantification of microalgal carbohydrates

Structural carbohydrates can constitute a large fraction of the dry weight of algal biomass and thus accurate identification and quantification is important for summative mass closure. Two limitations to the accurate characterization of microalgal carbohydrates are the lack of a robust analytical procedure to hydrolyze polymeric carbohydrates to their respective monomers and the subsequent identification and quantification of those monosaccharides. We address the second limitation, chromatographic separation of monosaccharides, here by identifying optimum conditions for the resolution of a synthetic mixture of 13 microalgae-specific monosaccharides, comprised of 8 neutral, 2 amino sugars, 2 uronic acids and 1 alditol (myo-inositol as an internal standard). The synthetic 13-carbohydrate mix showed incomplete resolution across 11 traditional high performance liquid chromatography (HPLC) methods, but showed improved resolution and accurate quantification using anion exchange chromatography (HPAEC) as well as alditol acetate derivatization followed by gas chromatography (for the neutral- and amino-sugars only). We demonstrate the application of monosaccharide quantification using optimized chromatography conditions after sulfuric acid analytical hydrolysis for three model algae strains and compare the quantification and complexity of monosaccharides in analytical hydrolysates relative to a typical terrestrial feedstock, sugarcane bagasse.

Strain, biochemistry, and cultivation-dependent measurement variability of algal biomass composition.

Accurate compositional analysis in biofuel feedstocks is imperative; the yields of individual components can define the economics of an entire process. In the nascent industry of algal biofuels and bioproducts, analytical methods that have been deemed acceptable for decades are suddenly critical for commercialization. We tackled the question of how the strain and biochemical makeup of algal cells affect chemical measurements. We selected a set of six procedures (two each for lipids, protein, and carbohydrates): three rapid fingerprinting methods and three advanced chromatography-based methods. All methods were used to measure the composition of 100 samples from three strains: Scenedesmus sp., Chlorella sp., and Nannochloropsis sp. The data presented point not only to species-specific discrepancies but also to cell biochemistry-related discrepancies. There are cases where two respective methods agree but the differences are often significant with over- or underestimation of up to 90%, likely due to chemical interferences with the rapid spectrophotometric measurements. We provide background on the chemistry of interfering reactions for the fingerprinting methods and conclude that for accurate compositional analysis of algae and process and mass balance closure, emphasis should be placed on unambiguous characterization using methods where individual components are measured independently.

Development of algae biorefinery concepts for biofuels and bioproducts; a perspective on process-compatible products and their impact on cost-reduction

Identifying and addressing critical improvements in biomass, bioproduct and biofuel productivity is a priority for the nascent algae-based bioeconomy. Economic and sustainability principles should guide these developing improvements and help to unravel the contentious water–food–energy–environment nexus that algae inhabit. Understanding the biochemistry of the storage carbon metabolism of algae to produce biofuels and bioproducts can bring to light the key barriers that currently limit the overall carbon efficiency and the photosynthetic efficiency, and ultimately guide productivity and commercial viability in the context of limiting resources. In the analysis reported here, we present different potential pathways for a conceptual algae biorefinery framework, with each pathway addressing one of the main identified barriers to future deployment. We highlight the molecular identification, in the form of an extensive literature review, of potential bioproducts that may be derived directly from both biomass and fractions produced through a conversion pathway, for three important commercially-relevant genera of algae, Scenedesmus, Chlorella and Nannochloropsis. We establish a relationship between each of the potential bioproducts, describe relevant conversion and extraction processes, and discuss market opportunities with values and sizes as they relate to commercial development of the products.

Genomic, Proteomic, and Biochemical Analyses of Oleaginous Mucor circinelloides: Evaluating Its Capability in Utilizing Cellulolytic Substrates for Lipid Production

Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory), 3 β-D-glucosidases (2 of them secretory) and 243 other glycoside hydrolase (GH) proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH) that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose) and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP) strain by introducing a CBH (e.g. CBHI) into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME) analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

Determination of Total Carbohydrates in Algal Biomass: Laboratory Analytical Procedure (LAP)

This procedure uses two-step sulfuric acid hydrolysis to hydrolyze the polymeric forms of carbohydrates in algal biomass into monomeric subunits. The monomers are then quantified by either HPLC or a suitable spectrophotometric method.

Oleaginicity of the yeast strain Saccharomyces cerevisiae D5A

BackgroundThe model yeast, Saccharomyces cerevisiae, is not known to be oleaginous. However, an industrial wild-type strain, D5A, was shown to accumulate over 20% storage lipids from glucose when growth is nitrogen-limited compared to no more than 7% lipid accumulation without nitrogen stress.Methods and resultsTo elucidate the mechanisms of S. cerevisiae D5A oleaginicity, we compared physiological and metabolic changes; as well as the transcriptional profiles of the oleaginous industrial strain, D5A, and a non-oleaginous laboratory strain, BY4741, under normal and nitrogen-limited conditions using analytic techniques and next-generation sequencing-based RNA-Seq transcriptomics. Transcriptional levels for genes associated with fatty acid biosynthesis, nitrogen metabolism, amino acid catabolism, as well as the pentose phosphate pathway and ethanol oxidation in central carbon (C) metabolism, were up-regulated in D5A during nitrogen deprivation. Despite increased carbon flux to lipids, most gene-encoding enzymes involved in triacylglycerol (TAG) assembly were expressed at similar levels regardless of the varying nitrogen concentrations in the growth media and strain backgrounds. Phospholipid turnover also contributed to TAG accumulation through increased precursor production with the down-regulation of subsequent phospholipid synthesis steps. Our results also demonstrated that nitrogen assimilation via the glutamate–glutamine pathway and amino acid metabolism, as well as the fluxes of carbon and reductants from central C metabolism, are integral to the general oleaginicity of D5A, which resulted in the enhanced lipid storage during nitrogen deprivation.ConclusionThis work demonstrated the disequilibrium and rebalance of carbon and nitrogen contribution to the accumulation of lipids in the oleaginous yeast S. cerevisiae D5A. Rather than TAG assembly from acyl groups, the major switches for the enhanced lipid accumulation of D5A (i.e., fatty acid biosynthesis) are the increases of cytosolic pools of acetyl-CoA and NADPH, as well as alternative nitrogen assimilation.