Herbivorous Fish Microbiome Adaptations to Sulfated Dietary Polysaccharides.

Marine herbivorous fish that feed primarily on macroalgae, such as those from the genus Kyphosus, are essential for maintaining coral health and abundance on tropical reefs. Here, deep metagenomic sequencing and assembly of gut compartment-specific samples from three sympatric, macroalgivorous Hawaiian kyphosid species have been used to connect host gut microbial taxa with predicted protein functional capacities likely to contribute to efficient macroalgal digestion. Bacterial community compositions, algal dietary sources, and predicted enzyme functionalities were analyzed in parallel for 16 metagenomes spanning the mid- and hindgut digestive regions of wild-caught fishes. Gene colocalization patterns of expanded carbohydrate (CAZy) and sulfatase (SulfAtlas) digestive enzyme families on assembled contigs were used to identify likely polysaccharide utilization locus associations and to visualize potential cooperative networks of extracellularly exported proteins targeting complex sulfated polysaccharides. These insights into the gut microbiota of herbivorous marine fish and their functional capabilities improve our understanding of the enzymes and microorganisms involved in digesting complex macroalgal sulfated polysaccharides. IMPORTANCE This work connects specific uncultured bacterial taxa with distinct polysaccharide digestion capabilities lacking in their marine vertebrate hosts, providing fresh insights into poorly understood processes for deconstructing complex sulfated polysaccharides and potential evolutionary mechanisms for microbial acquisition of expanded macroalgal utilization gene functions. Several thousand new marine-specific candidate enzyme sequences for polysaccharide utilization have been identified. These data provide foundational resources for future investigations into suppression of coral reef macroalgal overgrowth, fish host physiology, the use of macroalgal feedstocks in terrestrial and aquaculture animal feeds, and the bioconversion of macroalgae biomass into value-added commercial fuel and chemical products.

Single-filament imaging mass spectrometry lipidomics in Arthrospira platensis.

RATIONALE: Elucidating intra-organismal biochemical and lipid organization in photosynthetic biological cell factories of filamentous cyanobacteria, such as Arthrospira platensis (Spirulina), is important for tracking physiological response mechanisms during growth. Little is known about the filaments' biochemical organization and cellular structure and no label-free imaging techniques exist that provide molecular mapping. METHODS: We applied ultrahigh-resolution mass spectrometry (MS) with matrix-assisted laser desorption ionization (MALDI) imaging to immobilized Spirulina filaments to investigate the localization of lipids across distinct physiological regions. We optimized matrix selection and deposition methods with the goal of facilitating high spatial, and intra-filament, resolution using untargeted multivariate statistical spectral deconvolution across MS pixels. RESULTS: Our results demonstrate an improved two-step matrix application with an optimized procedure for intra-organismal lipid profiling to improve analyte sensitivity and achieve higher spatial resolution. We evaluate several conventional matrices, namely 2,5-dihydroxybenzoic acid (DHB), superDHB (sDHB), 1,5-diaminonaphthalene (DAN), and a 50:50 mix of DHB and sDHB, and compare delineation and pixel-based elucidation of intra-filament lipidomics. We identified a total of 1626 features that could be putatively assigned a lipid-like formula based on database query and 46 unique features, with associated lipid assignments that were significantly distinct in their intra-filament location. CONCLUSIONS: MALDI imaging MS with untargeted statistical spectral deconvolution was used to visualize intra-filament lipidomics organization in Spirulina filaments. Improvements in matrix deposition, including sequential sublimation and pneumatic spraying, increased signal abundance at high spatial resolution and allowed for identification of distinct lipid composition regions. This work outlines a methodology that may be used for micro-ecological untargeted molecular phenotyping.

A Unified Modeling Framework to Advance Biofuel Production from Microalgae.

Modeling efforts to understand the financial implications of microalgal biofuels often assume a static basis for microalgae biomass composition and cost, which has constrained cultivation and downstream conversion process design and limited in-depth understanding of their interdependencies. For this work, a dynamic biological cultivation model was integrated with thermo-chemical/biological unit process models for downstream biorefineries to increase modeling fidelity, to provide mechanistic links among unit operations, and to quantify minimum product selling prices of biofuels via techno-economic analysis. Variability in design, cultivation, and conversion parameters were characterized through Monte Carlo simulation, and sensitivity analyses were conducted to identify key cost and fuel yield drivers. Cultivating biomass to achieve the minimum biomass selling price or to achieve maximum lipid content were shown to lead to suboptimal fuel production costs. Depending on biomass composition, both hydrothermal liquefaction and a biochemical fractionation process (combined algal processing) were shown to have advantageous minimum product selling prices, which supports continued investment in multiple conversion pathways. Ultimately, this work demonstrates a clear need to leverage integrated modeling platforms to advance microalgae biofuel systems as a whole, and specific recommendations are made for the prioritization of research and development pathways to achieve economical biofuel production from microalgae.

MBTH: A novel approach to rapid, spectrophotometric quantitation of total algal carbohydrates.

A high-throughput and robust application of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method was developed for carbohydrate determination in microalgae. The traditional phenol-sulfuric acid method to quantify carbohydrates is strongly affected by algal biochemical components and exhibits a highly variable response to microalgal monosaccharides. We present a novel use of the MBTH method to accurately quantify carbohydrates in hydrolyzate after acid hydrolysis of algal biomass, without a need for neutralization. The MBTH method demonstrated consistent and sensitive quantitation of algae-specific monosaccharides down to 5 µg mL-1 without interference from other algae acidic hydrolyzate components.

Genome-scale metabolic reconstructions and theoretical investigation of methane conversion in Methylomicrobium buryatense strain 5G(B1).

BACKGROUND: Methane-utilizing bacteria (methanotrophs) are capable of growth on methane and are attractive systems for bio-catalysis. However, the application of natural methanotrophic strains to large-scale production of value-added chemicals/biofuels requires a number of physiological and genetic alterations. An accurate metabolic model coupled with flux balance analysis can provide a solid interpretative framework for experimental data analyses and integration. RESULTS: A stoichiometric flux balance model of Methylomicrobium buryatense strain 5G(B1) was constructed and used for evaluating metabolic engineering strategies for biofuels and chemical production with a methanotrophic bacterium as the catalytic platform. The initial metabolic reconstruction was based on whole-genome predictions. Each metabolic step was manually verified, gapfilled, and modified in accordance with genome-wide expression data. The final model incorporates a total of 841 reactions (in 167 metabolic pathways). Of these, up to 400 reactions were recruited to produce 118 intracellular metabolites. The flux balance simulations suggest that only the transfer of electrons from methanol oxidation to methane oxidation steps can support measured growth and methane/oxygen consumption parameters, while the scenario employing NADH as a possible source of electrons for particulate methane monooxygenase cannot. Direct coupling between methane oxidation and methanol oxidation accounts for most of the membrane-associated methane monooxygenase activity. However the best fit to experimental results is achieved only after assuming that the efficiency of direct coupling depends on growth conditions and additional NADH input (about 0.1-0.2 mol of incremental NADH per one mol of methane oxidized). The additional input is proposed to cover loss of electrons through inefficiency and to sustain methane oxidation at perturbations or support uphill electron transfer. Finally, the model was used for testing the carbon conversion efficiency of different pathways for C1-utilization, including different variants of the ribulose monophosphate pathway and the serine cycle. CONCLUSION: We demonstrate that the metabolic model can provide an effective tool for predicting metabolic parameters for different nutrients and genetic perturbations, and as such, should be valuable for metabolic engineering of the central metabolism of M. buryatense strains.

Profiling Chlamydomonas metabolism under dark, anoxic H2-producing conditions using a combined proteomic, transcriptomic, and metabolomic approach.

Chlamydomonas reinhardtii is well adapted to survive under different environmental conditions due to the unique flexibility of its metabolism. Here we report metabolic pathways that are active during acclimation to anoxia, but were previously not thoroughly studied under dark, anoxic H2-producing conditions in this model green alga. Proteomic analyses, using 2D-differential in-gel electrophoresis in combination with shotgun mass fingerprinting, revealed increased levels of proteins involved in the glycolytic pathway downstream of 3-phosphoglycerate, the glyoxylate pathway, and steps of the tricarboxylic acid (TCA) reactions. Upregulation of the enzyme, isocitrate lyase (ICL), was observed, which was accompanied by increased intracellular succinate levels, suggesting the functioning of glyoxylate pathway reactions. The ICL-inhibitor study revealed presence of reverse TCA reactions under these conditions. Contributions of the serine-isocitrate lyase pathway, glycine cleavage system, and c1-THF/serine hydroxymethyltransferase pathway in the acclimation to dark anoxia were found. We also observed increased levels of amino acids (AAs) suggesting nitrogen reorganization in the form of de novo AA biosynthesis during anoxia. Overall, novel routes for reductant utilization, in combination with redistribution of carbon and nitrogen, are used by this alga during acclimation to O2 deprivation in the dark.

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.

High-Resolution Lipidomics Reveals Influence of Biomass and Pretreatment Process on the Composition of Extracted Algae Oils As Feedstock for Sustainable Aviation Fuels.

The increasing demand for sustainable aviation fuel (SAF) creates a need for innovative biomass and lipid sources with compositions that are compatible with refineries. Algae-derived oils present an opportunity to supply a process-compatible lipid feedstock at yields higher than those of conventional oilseed crops. With few documented reports on chemical composition, the process readiness remains elusive. We present data on extraction efficiency, yield, and purity of lipids from algae with and without the application of a low-concentration sulfuric acid pretreatment of the biomass. The pretreatment process increased the oil yield and positively impacted the quality of the extracted oils. Results from fatty acid and lipidomics analysis revealed that the low-lipid biomass sources extracted 70-80% of the available lipids, and the non-fatty acid co-extractants exceeded 40% of the extracted oils. For a high-lipid algae sample, derived from a genetically engineered strain, we show >90% extraction yield with >85% FAME purity. This work provides insights into the composition of algae-derived oils and quality metrics that are essential to determining the viability of lipid hydroprocessing to SAF.

Central transcriptional regulator controls photosynthetic growth and carbon storage in response to high light.

Carbon capture and biochemical storage are some of the primary drivers of photosynthetic yield and productivity. To elucidate the mechanisms governing carbon allocation, we designed a photosynthetic light response test system for genetic and metabolic carbon assimilation tracking, using microalgae as simplified plant models. The systems biology mapping of high light-responsive photophysiology and carbon utilization dynamics between two variants of the same Picochlorum celeri species, TG1 and TG2 elucidated metabolic bottlenecks and transport rates of intermediates using instationary 13C-fluxomics. Simultaneous global gene expression dynamics showed 73% of the annotated genes responding within one hour, elucidating a singular, diel-responsive transcription factor, closely related to the CCA1/LHY clock genes in plants, with significantly altered expression in TG2. Transgenic P. celeri TG1 cells expressing the TG2 CCA1/LHY gene, showed 15% increase in growth rates and 25% increase in storage carbohydrate content, supporting a coordinating regulatory function for a single transcription factor.

Enrichable consortia of microbial symbionts degrade macroalgal polysaccharides in Kyphosus fish.

Coastal herbivorous fishes consume macroalgae, which is then degraded by microbes along their digestive tract. However, there is scarce genomic information about the microbiota that perform this degradation. This study explores the potential of Kyphosus gastrointestinal microbial symbionts to collaboratively degrade and ferment polysaccharides from red, green, and brown macroalgae through in silico study of carbohydrate-active enzyme and sulfatase sequences. Recovery of metagenome-assembled genomes (MAGs) from previously described Kyphosus gut metagenomes and newly sequenced bioreactor enrichments reveals differences in enzymatic capabilities between the major microbial taxa in Kyphosus guts. The most versatile of the recovered MAGs were from the Bacteroidota phylum, whose MAGs house enzyme collections able to decompose a variety of algal polysaccharides. Unique enzymes and predicted degradative capacities of genomes from the Bacillota (genus Vallitalea) and Verrucomicrobiota (order Kiritimatiellales) highlight the importance of metabolic contributions from multiple phyla to broaden polysaccharide degradation capabilities. Few genomes contain the required enzymes to fully degrade any complex sulfated algal polysaccharide alone. The distribution of suitable enzymes between MAGs originating from different taxa, along with the widespread detection of signal peptides in candidate enzymes, is consistent with cooperative extracellular degradation of these carbohydrates. This study leverages genomic evidence to reveal an untapped diversity at the enzyme and strain level among Kyphosus symbionts and their contributions to macroalgae decomposition. Bioreactor enrichments provide a genomic foundation for degradative and fermentative processes central to translating the knowledge gained from this system to the aquaculture and bioenergy sectors.IMPORTANCESeaweed has long been considered a promising source of sustainable biomass for bioenergy and aquaculture feed, but scalable industrial methods for decomposing terrestrial compounds can struggle to break down seaweed polysaccharides efficiently due to their unique sulfated structures. Fish of the genus Kyphosus feed on seaweed by leveraging gastrointestinal bacteria to degrade algal polysaccharides into simple sugars. This study reconstructs metagenome-assembled genomes for these gastrointestinal bacteria to enhance our understanding of herbivorous fish digestion and fermentation of algal sugars. Investigations at the gene level identify Kyphosus guts as an untapped source of seaweed-degrading enzymes ripe for further characterization. These discoveries set the stage for future work incorporating marine enzymes and microbial communities in the industrial degradation of algal polysaccharides.

New method for discovery of starch phenotypes in growing microalgal colonies.

To identify algal strains with altered starch metabolism from a large pool of candidates of growing algal colonies, we have developed a novel, high-throughput screening tool by combining gaseous bleaching of replica transferred colonies and subsequent iodine staining to visualize starch. Screening of healthy growing colonies of microalgae has not been possible previously because high levels of chlorophyll make the detection of starch with an iodine stain impossible. We demonstrated that chlorine dioxide (ClO(2)) removes essentially all chlorophyll from the colonies and enables high-throughput screening of, for example, a population of mutagenized cells or a culture collection isolated in a bioprospecting project.

Tropical Red Macroalgae Cultivation with a Focus on Compositional Analysis.

To create carbon efficient sources of bioenergy feedstocks and feedstuff for aquaculture and terrestrial livestock, it is critical to develop and commercialize the most efficient seaweed cultivation approach with a sustainable nutrient input supply. Here, we present data for a novel, onshore tropical macroalgae cultivation system, based on influent deep seawater as the nutrient and carbon sources. Two red algal species were selected, Agardhiella subulata and Halymenia hawaiiana, as the basis for growth optimization. Highest productivity in small-scale cultivation was demonstrated with A. subulata in the 10% deep seawater (64.7 µg N L-1) treatment, growing at up to 26% specific growth rate day-1 with highest yields observed at 247.5 g m-2 day-1 fresh weight. The highest yields for H. hawaiiana were measured with the addition of 10% deep seawater up to 8.8% specific growth rate day-1 and yields at 63.3 g fresh weight m-2 day-1 equivalent. Biomass should be culled weekly or biweekly to avoid density limitations, which likely contributed to a decrease in SGR over time. With a measured 30-40% carbon content of the ash-free dry weight (20-30% of the dry weight) biomass, this translates to an almost 1:1 CO2 capture to biomass ratio. The compositional fingerprint of the high carbohydrate content of both Agardhiella and Halymenia makes for an attractive feedstock for downstream biorefinery applications. By focusing on scaling and optimizing seaweed farming technologies for large-scale onshore farms, the opportunities for yield potential, adaptability to cultivation conditions, and meeting global sustainability goals through novel, carbon-negative biomass sources such as seaweed can be realized.

Enrichable consortia of microbial symbionts degrade macroalgal polysaccharides in Kyphosus fish.

Coastal herbivorous fishes consume macroalgae, which is then degraded by microbes along their digestive tract. However, there is scarce foundational genomic work on the microbiota that perform this degradation. This study explores the potential of Kyphosus gastrointestinal microbial symbionts to collaboratively degrade and ferment polysaccharides from red, green, and brown macroalgae through in silico study of carbohydrate-active enzyme and sulfatase sequences. Recovery of metagenome-assembled genomes (MAGs) reveals differences in enzymatic capabilities between the major microbial taxa in Kyphosus guts. The most versatile of the recovered MAGs were from the Bacteroidota phylum, whose MAGs house enzymes able to decompose a variety of algal polysaccharides. Unique enzymes and predicted degradative capacities of genomes from the Bacillota (genus Vallitalea) and Verrucomicrobiota (order Kiritimatiellales) suggest the potential for microbial transfer between marine sediment and Kyphosus digestive tracts. Few genomes contain the required enzymes to fully degrade any complex sulfated algal polysaccharide alone. The distribution of suitable enzymes between MAGs originating from different taxa, along with the widespread detection of signal peptides in candidate enzymes, is consistent with cooperative extracellular degradation of these carbohydrates. This study leverages genomic evidence to reveal an untapped diversity at the enzyme and strain level among Kyphosus symbionts and their contributions to macroalgae decomposition. Bioreactor enrichments provide a genomic foundation for degradative and fermentative processes central to translating the knowledge gained from this system to the aquaculture and bioenergy sectors.

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.

Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by in situ transesterification.

In the context of algal biofuels, lipids, or better aliphatic chains of the fatty acids, are perhaps the most important constituents of algal biomass. Accurate quantification of lipids and their respective fuel yield is crucial for comparison of algal strains and growth conditions and for process monitoring. As an alternative to traditional solvent-based lipid extraction procedures, we have developed a robust whole-biomass in situ transesterification procedure for quantification of algal lipids (as fatty acid methyl esters, FAMEs) that (a) can be carried out on a small scale (using 4-7 mg of biomass), (b) is applicable to a range of different species, (c) consists of a single-step reaction, (d) is robust over a range of different temperature and time combinations, and (e) tolerant to at least 50% water in the biomass. Unlike gravimetric lipid quantification, which can over- or underestimate the lipid content, whole biomass transesterification reflects the true potential fuel yield of algal biomass. We report here on the comparison of the yield of FAMEs by using different catalysts and catalyst combinations, with the acid catalyst HCl providing a consistently high level of conversion of fatty acids with a precision of 1.9% relative standard deviation. We investigate the influence of reaction time, temperature, and biomass water content on the measured FAME content and profile for 4 different samples of algae (replete and deplete Chlorella vulgaris, replete Phaeodactylum tricornutum, and replete Nannochloropsis sp.). We conclude by demonstrating a full mass balance closure of all fatty acids around a traditional lipid extraction process.

Fine scale transitions of the microbiota and metabolome along the gastrointestinal tract of herbivorous fishes.

BACKGROUND: Gut microorganisms aid in the digestion of food by providing exogenous metabolic pathways to break down organic compounds. An integration of longitudinal microbial and chemical data is necessary to illuminate how gut microorganisms supplement the energetic and nutritional requirements of animals. Although mammalian gut systems are well-studied in this capacity, the role of microbes in the breakdown and utilization of recalcitrant marine macroalgae in herbivorous fish is relatively understudied and an emerging priority for bioproduct extraction. Here we use a comprehensive survey of the marine herbivorous fish gut microbial ecosystem via parallel 16S rRNA gene amplicon profiling (microbiota) and untargeted tandem mass spectrometry (metabolomes) to demonstrate consistent transitions among 8 gut subsections across five fish of the genus of Kyphosus. RESULTS: Integration of microbial phylogenetic and chemical diversity data reveals that microbial communities and metabolomes covaried and differentiated continuously from stomach to hindgut, with the midgut containing multiple distinct and previously uncharacterized microenvironments and a distinct hindgut community dominated by obligate anaerobes. This differentiation was driven primarily by anaerobic gut endosymbionts of the classes Bacteroidia and Clostridia changing in concert with bile acids, small peptides, and phospholipids: bile acid deconjugation associated with early midgut microbiota, small peptide production associated with midgut microbiota, and phospholipid production associated with hindgut microbiota. CONCLUSIONS: The combination of microbial and untargeted metabolomic data at high spatial resolution provides a new view of the diverse fish gut microenvironment and serves as a foundation to understand functional partitioning of microbial activities that contribute to the digestion of complex macroalgae in herbivorous marine fish.

Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains.

The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O(2) evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O(2) evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.

Sustainable Seaweed Biotechnology Solutions for Carbon Capture, Composition, and Deconstruction.

Seaweeds or macroalgae are attractive candidates for carbon capture, while also supplying a sustainable photosynthetic bioenergy feedstock, thanks to their cultivation potential in offshore marine farms. Seaweed cultivation requires minimal external nutrient requirements and allows for year-round production of biomass. Despite this potential, there remain significant challenges associated with realizing large-scale, sustainable agronomics, as well as in the development of an efficient biomass deconstruction and conversion platform to fuels and products. Recent biotechnology progress in the identification of enzymatic deconstruction pathways, tailored to complex polymers in seaweeds, opens up opportunities for more complete utilization of seaweed biomass components. Effective, scalable, and economically viable conversion processes tailored to seaweed are discussed and gaps are identified for yield and efficiency improvements.

Draft Genome Sequence of the Biofuel-Relevant Microalga Desmodesmus armatus.

A draft genome of 906 scaffolds of 115.8 Mb was assembled for Desmodesmus armatus, a diploid, lipid- and storage carbohydrate-accumulating microalga proven relevant for large-scale, outdoor cultivation, and serves as a model alga platform for improving photosynthetic efficiency and carbon assimilation for next-generation bioenergy production.

Total Protein Analysis in Algae via Bulk Amino Acid Detection: Optimization of Amino Acid Derivatization after Hydrolysis with O-Phthalaldehyde 3-Mercaptopropionic Acid (OPA-3MPA).

The analysis of protein in algal biomass is one of the most critical areas of commercial development of algae characterization for nutritional or other high value applications. A new rapid and accurate method is required that can be widely implemented and that is free from interferences from the complex algal biomass matrix. We developed a simple spectrophotometric method for primary amino acid quantification bulk measurement in an acid hydrolyzed algal biomass preparation, as an alternative to the more labor-intensive amino HPLC acid analysis or less specific nitrogen-to-protein conversion. We have validated an O-phthalaldehyde (OPA)-based derivatization method, showing accurate and linear quantification for standard reference amino acids as well as mixtures, mimicking the amino acid complexity found in algal biomass. The presence of interferences that may be derived from the complex biomass biochemical composition was tested during the method validation phase. We document the application of a novel method of OPA derivatization with 3-mercaptopropionic acid (3MPA) to determine the total amino acid content of harvested algal biomass collected from different, controlled cultivation conditions and demonstrated a within 10% accuracy against a reference measurement of amino acid content in at least 4 species and 10 algal biomass samples, across early, mid, and late-stages of cultivation.