Coordinated proteome change precedes cell lysis and death in a mat-forming cyanobacterium.

Cyanobacteria form dense multicellular communities that experience transient conditions in terms of access to light and oxygen. These systems are productive but also undergo substantial biomass turnover through cell death, supplementing heightened heterotrophic respiration. Here we use metagenomics and metaproteomics to survey the molecular response of a mat-forming cyanobacterium undergoing mass cell lysis after exposure to dark and anoxic conditions. A lack of evidence for viral, bacterial, or eukaryotic antagonism contradicts commonly held beliefs on the causative agent for cyanobacterial death during dense growth. Instead, proteogenomics data indicated that lysis likely resulted from a genetically programmed response triggered by a failure to maintain osmotic pressure in the wake of severe energy limitation. Cyanobacterial DNA was rapidly degraded, yet cyanobacterial proteins remained abundant. A subset of proteins, including enzymes involved in amino acid metabolism, peptidases, toxin-antitoxin systems, and a potentially self-targeting CRISPR-Cas system, were upregulated upon lysis, indicating possible involvement in the programmed cell death response. We propose this natural form of cell death could provide new pathways for controlling harmful algal blooms and for sustainable bioproduct production.

Autofermentation of alkaline cyanobacterial biomass to enable biorefinery approach.

BACKGROUND: Carbon capture using alkaliphilic cyanobacteria can be an energy-efficient and environmentally friendly process for producing bioenergy and bioproducts. The inefficiency of current harvesting and downstream processes, however, hinders large-scale feasibility. The high alkalinity of the biomass also introduces extra challenges, such as potential corrosion, inhibitory effects, or contamination of the final products. Thus, it is critical to identify low cost and energy-efficient downstream processes. RESULTS: Autofermentation was investigated as an energy-efficient and low-cost biomass pre-treatment method to reduce pH to levels suitable for downstream processes, enabling the conversion of cyanobacterial biomass into hydrogen and organic acids using cyanobacteria's own fermentative pathways. Temperature, initial biomass concentration, and oxygen presence were found to affect yield and distribution of organic acids. Autofermentation of alkaline cyanobacterial biomass was found to be a viable approach to produce hydrogen and organic acids simultaneously, while enabling the successful conversion of biomass to biogas. Between 5.8 and 60% of the initial carbon was converted into organic acids, 8.7-25% was obtained as soluble protein, and 16-72% stayed in the biomass. Interestingly, we found that extensive dewatering is not needed to effectively process the alkaline cyanobacterial biomass. Using natural settling as the only harvesting and dewatering method resulted in a slurry with relatively low biomass concentration. Nevertheless, autofermentation of this slurry led to the maximum total organic acid yield (60% C mol/C mol biomass) and hydrogen yield (326.1 µmol/g AFDM). CONCLUSION: Autofermentation is a simple, but highly effective pretreatment that can play a significant role within a cyanobacterial-based biorefinery platform by enabling the conversion of alkaline cyanobacterial biomass into organic acids, hydrogen, and methane via anaerobic digestion without the addition of energy or chemicals.

Nutrient management and medium reuse for cultivation of a cyanobacterial consortium at high pH and alkalinity.

Alkaliphilic cyanobacteria have gained significant interest due to their robustness, high productivity, and ability to convert CO2 into bioenergy and other high value products. Effective nutrient management, such as re-use of spent medium, will be essential to realize sustainable applications with minimal environmental impacts. In this study, we determined the solubility and uptake of nutrients by an alkaliphilic cyanobacterial consortium grown at high pH and alkalinity. Except for Mg, Ca, Co, and Fe, all nutrients are in fully soluble form. The cyanobacterial consortium grew well without any inhibition and an overall productivity of 0.15 g L-1 d-1 (AFDW) was achieved. Quantification of nutrient uptake during growth resulted in the empirical formula CH1.81N0.17O0.20P0.013S0.009 for the consortium biomass. We showed that spent medium can be reused for at least five growth/harvest cycles. After an adaptation period, the cyanobacterial consortium fully acclimatized to the spent medium, resulting in complete restoration of biomass productivity.

Pilot-scale outdoor trial of a cyanobacterial consortium at pH 11 in a photobioreactor at high latitude.

The biomass of microalgae and cyanobacteria yields a variety of products. Outdoor pilot plant trials typically grow a single species at circumneutral pH and provide CO2 by gas sparging. Here a cyanobacterial consortium was grown at high pH (beyond 11) and high dissolved carbonate concentrations (0.5 M) in an outdoor 1,150 L tubular photobioreactor for 130 days in Calgary, Canada. The aim was to assess the productivity and robustness of the consortium. Importantly, the system was designed to enable future integration of air capture of CO2. Productivity was between 3.1 and 5.8 g ash-free dry weight per square metre per day, depending on biomass density and month. 16S rRNA amplicon sequencing showed that cyanobacterium Candidatus "Phormidium alkaliphilum" made up 80% of the consortium. The consortium displayed robust growth and adapted to environmental conditions. Bicarbonate uptake pushed medium pH past 11, demonstrating the ability to achieve CO2 delivery by air capture.

Proteome and strain analysis of cyanobacterium Candidatus "Phormidium alkaliphilum" reveals traits for success in biotechnology.

Cyanobacteria encompass a diverse group of photoautotrophic bacteria with important roles in nature and biotechnology. Here we characterized Candidatus "Phormidium alkaliphilum," an abundant member in alkaline soda lake microbial communities globally. The complete, circular whole-genome sequence of Ca. "P. alkaliphilum" was obtained using combined Nanopore and Illumina sequencing of a Ca. "P. alkaliphilum" consortium. Strain-level diversity of Ca. "P. alkaliphilum" was shown to contribute to photobioreactor robustness under different operational conditions. Comparative genomics of closely related species showed that adaptation to high pH was not attributed to specific genes. Proteomics at high and low pH showed only minimal changes in gene expression, but higher productivity in high pH. Diverse photosystem antennae proteins, and high-affinity terminal oxidase, compared with other soda lake cyanobacteria, appear to contribute to the success of Ca. "P. alkaliphilum" in photobioreactors and biotechnology applications.

Quantification of Lipid Content in Oleaginous Biomass Using Thermogravimetry.

Laboratory analytical techniques employed for triglyceride quantification in oleaginous biomass (e.g., microalgae and oilseeds) involve multiple steps and typically require use of volatile organic solvents. Here we describe a single-step approach for measurement of triglycerides using thermogravimetry (TG). We have observed that triglycerides undergo complete volatilization over a narrow temperature interval of 370-450 °C, with negligible solid residue under inert atmosphere, whereas other constituents of oleaginous biomass (such as proteins and carbohydrates) primarily degrade below 350 °C. As a result, triglyceride content of biomass can be estimated using TG by determining the mass loss of the sample in the temperature interval of 370-450 °C.

Direct capture and conversion of CO2 from air by growing a cyanobacterial consortium at pH up to 11.2.

Bioenergy with carbon capture and storage (BECCS) is recognized as a potential negative emission technology, needed to keep global warming within safe limits. With current technologies, large-scale implementation of BECCS would compromise food production. Bioenergy derived from phototrophic microorganisms, with direct capture of CO2 from air, could overcome this challenge and become a sustainable way to realize BECCS. Here we present an alkaline capture and conversion system that combines high atmospheric CO2 transfer rates with high and robust phototrophic biomass productivity (15.2 ± 1.0 g/m 2 /d). The system is based on a cyanobacterial consortium, that grows at high alkalinity (0.5 mol/L) and a pH swing between 10.4 and 11.2 during growth and harvest cycles.

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.