Mixed and membrane-separated culturing of synthetic cyanobacteria-yeast consortia reveals metabolic cross-talk mimicking natural cyanolichens.

Metabolite exchange mediates crucial interactions in microbial communities, significantly impacting global carbon and nitrogen cycling. Understanding these chemically-mediated interactions is essential for elucidating natural community functions and developing engineered synthetic communities. This study investigated membrane-separated bioreactors (mBRs) as a novel tool to identify transient metabolites and their producers/consumers in mixed microbial communities. We compared three co-culture methods (direct mixed, 2-chamber mBR, and 3-chamber mBR) to grow a synthetic binary community of the cyanobacterium Synechococcus elongatus PCC 7942 and the fungus Rhodotorula toruloides NBRC 0880, as well as axenic S. elongatus. Despite not being natural lichen constituents, these organisms exhibited interactions resembling those in cyanolichens. S. elongatus fixed CO2 into sugars as the primary shared metabolite, while R. toruloides secreted various biochemicals, predominantly sugar alcohols, mirroring the metabolite exchange observed in natural lichens. The mBR systems successfully captured metabolite gradients and revealed rapidly consumed compounds, including TCA cycle intermediates and amino acids. Our approach demonstrated that the 2-chamber mBR optimally balanced metabolite exchange and growth dynamics. This study provides insights into cross-species metabolic interactions and presents a valuable tool for investigating and engineering synthetic microbial communities with potential applications in biotechnology and environmental science.

PTM-Psi: A python package to facilitate the computational investigation of post-translational modification on protein structures and their impacts on dynamics and functions.

Post-translational modification (PTM) of a protein occurs after it has been synthesized from its genetic template, and involves chemical modifications of the protein's specific amino acid residues. Despite of the central role played by PTM in regulating molecular interactions, particularly those driven by reversible redox reactions, it remains challenging to interpret PTMs in terms of protein dynamics and function because there are numerous combinatorially enormous means for modifying amino acids in response to changes in the protein environment. In this study, we provide a workflow that allows users to interpret how perturbations caused by PTMs affect a protein's properties, dynamics, and interactions with its binding partners based on inferred or experimentally determined protein structure. This Python-based workflow, called PTM-Psi, integrates several established open-source software packages, thereby enabling the user to infer protein structure from sequence, develop force fields for non-standard amino acids using quantum mechanics, calculate free energy perturbations through molecular dynamics simulations, and score the bound complexes via docking algorithms. Using the S-nitrosylation of several cysteines on the GAP2 protein as an example, we demonstrated the utility of PTM-Psi for interpreting sequence-structure-function relationships derived from thiol redox proteomics data. We demonstrate that the S-nitrosylated cysteine that is exposed to the solvent indirectly affects the catalytic reaction of another buried cysteine over a distance in GAP2 protein through the movement of the two ligands. Our workflow tracks the PTMs on residues that are responsive to changes in the redox environment and lays the foundation for the automation of molecular and systems biology modeling.

Diploid genomic architecture of Nitzschia inconspicua, an elite biomass production diatom.

A near-complete diploid nuclear genome and accompanying circular mitochondrial and chloroplast genomes have been assembled from the elite commercial diatom species Nitzschia inconspicua. The 50 Mbp haploid size of the nuclear genome is nearly double that of model diatom Phaeodactylum tricornutum, but 30% smaller than closer relative Fragilariopsis cylindrus. Diploid assembly, which was facilitated by low levels of allelic heterozygosity (2.7%), included 14 candidate chromosome pairs composed of long, syntenic contigs, covering 93% of the total assembly. Telomeric ends were capped with an unusual 12-mer, G-rich, degenerate repeat sequence. Predicted proteins were highly enriched in strain-specific marker domains associated with cell-surface adhesion, biofilm formation, and raphe system gliding motility. Expanded species-specific families of carbonic anhydrases suggest potential enhancement of carbon concentration efficiency, and duplicated glycolysis and fatty acid synthesis pathways across cytosolic and organellar compartments may enhance peak metabolic output, contributing to competitive success over other organisms in mixed cultures. The N. inconspicua genome delivers a robust new reference for future functional and transcriptomic studies to illuminate the physiology of benthic pennate diatoms and harness their unique adaptations to support commercial algae biomass and bioproduct production.

Metals and methylotrophy: Via global gene expression studies.

A number of minerals, such as copper, cobalt, and rare earth elements (REE), are essential modulators of microbial one-carbon metabolism. This chapter provides an overview of the gene expression study design and analysis protocols for uncovering REE-induced changes in methylotrophic bacteria. By interrogating relationships and differences in total gene expression induced by mineral micronutrients, a deeper understanding of gene regulation at a systems scale can be gained. With careful design and execution of RNA-sequencing experiments, thorough processing and assessment of read quality can be utilized to assess and adjust for possible biases. By ensuring only quality data are utilized in downstream processes, differential gene expression, overrepresented analyses, and gene-set enrichment analyses provide reliable and reproducible representation of pathways and functions which are being affected by changes in environmental conditions.

Influence of polymer type and carbon nanotube properties on carbon nanotube/polymer nanocomposite biodegradation.

The interaction of anaerobic microorganisms with carbon nanotube/polymer nanocomposites (CNT/PNC) will play a major role in determining their persistence and environmental fate at the end of consumer use when these nano-enabled materials enter landfills and encounter wastewater. Motivated by the need to understand how different parameters (i.e., polymer type, microbial phenotype, CNT characteristics) influence CNT/PNC biodegradation rates, we have used volumetric biogas measurements and kinetic modeling to study biodegradation as a function of polymer type and CNT properties. In one set of experiments, oxidized multiwall carbon nanotubes (O-MWCNTs) with a range of CNT loadings 0-5% w/w were incorporated into poly-ε-caprolactone (PCL) and polyhydroxyalkanoates (PHA) matrices and subjected to biodegradation by an anaerobic microbial community. For each CNT/PNC, complete polymer biodegradation was ultimately observed, although the rate of biodegradation was inhibited above certain critical CNT loadings dependent upon the polymer type. Higher loadings of pristine MWCNTs were needed to decrease the rate of polymer biodegradation compared to O-MWCNTs, an effect ascribed principally to differences in CNT dispersion within the polymer matrices. Above certain CNT loadings, a CNT mat of similar shape to the initial PNC was formed after polymer biodegradation, while below this threshold, CNT aggregates fragmented in the media. In situations where biodegradation was rapid, methanogen growth was disproportionately inhibited compared to the overall microbial community. Analysis of the results obtained from this study indicates that the inhibitory effect of CNTs on polymer biodegradation rate is greatest under conditions (i.e., polymer type, microbial phenotype, CNT dispersion) where biodegradation of the neat polymer is slowest. This new insight provides a means to predict the environmental fate, persistence, and transformations of CNT-enabled polymer materials.

Production of lipid-containing algal-bacterial polyculture in wastewater and biomethanation of lipid extracted residues: Enhancing methane yield through hydrothermal pretreatment and relieving solvent toxicity through co-digestion.

The feasibility of generating a lipid-containing algal-bacterial polyculture biomass in municipal primary wastewater and enhancing biomethanation of lipid-extracted algal residues (LEA) through hydrothermal pretreatment and co-digestion with sewage sludge (SS) was investigated. In high-rate algal ponds, the polyculture of native algal and bacteria species demonstrated a monthly average net and gross biomass productivity of 30 ±â€¯3 and 36 ±â€¯3 gAFDW m-2 day-1 (summer season). The algal community was dominated by Micractinium sp. followed by Scenedesmus sp., Chlorella sp., pennate diatoms and Chlamydomonas sp. The polyculture metabolic activities resulted in average reductions of wastewater volatile suspended solids (VSS), carbonaceous soluble biochemical oxygen demand (csBOD5) and total nitrogen (Ntotal) of 63 ±â€¯18%, 98 ±â€¯1% and 76 ±â€¯21%, respectively. Harvested biomass contained nearly 23% lipid content and an extracted blend of fatty acid methyl esters satisfied the ASTM D6751 standard for biodiesel. Anaerobic digestion of lipid extracted algal residues (LEA) demonstrated long lag-phase in methane production of 17 days and ultimate methane yield of 296 ±â€¯2 mL/gVS (or ~50% of theoretical), likely because to its limited biodegradability and toxicity due to presence of the residual solvent (hexane). Hydrothermal pretreatment increased the ultimate methane yield and production rate by 15-30% but did not mitigate solvent toxicity effects completely leading to less substantial improvement in energy output of 5-20% and diminished Net Energy Ratio (NER < 1). In contrast, co-digestion of LEA with sewage sludge (10% to 90% ratio) was found to minimize solvent toxicity and improve methane yield enhancing the energy output ~4-fold, compared to using LEA as a single substrate, and advancing NER to 4.2.

Synergistic co-digestion of wastewater grown algae-bacteria polyculture biomass and cellulose to optimize carbon-to-nitrogen ratio and application of kinetic models to predict anaerobic digestion energy balance.

This study investigated enhancing methane production from algal-bacteria biomass by adjusting the C/N ratio through co-digestion with a nitrogen-poor co-substrate - cellulose. A biomethane potential test was used to determine cumulative biogas and methane production for pure and co-digested substrates. Four kinetic models were evaluated for their accuracy describing experimental data. These models were used to estimate the total energy output and net energy ratio (NER) for a scaled AD system. Increasing the algal C/N ratio from 5.7 to 20-30 (optimal algae:cellulose feedstock ratios of 35%:65% and 20%:80%) improved the ultimate methane yield by >10% and the first ten days production by >100%. The modified Gompertz kinetic model demonstrated highest accuracy, predicting that co-digestion improved methane production by reducing the time-lag by ∼50% and increasing rate by ∼35%. The synergistic effects increase the AD system energy efficiency and NER by 30-45%, suggesting potential for substantial enhancements from co-digestion at scale.

Draft Genome Sequence of Aspergillus oryzae ATCC 12892.

The draft genome sequence of Aspergillus oryzae ATCC 12892 is presented here. A. oryzae produces 3-nitropropionic acid, which has been investigated with regard to understanding the biosynthesis of nitroorganic compounds.

Conversion of stranded waste-stream carbon and nutrients into value-added products via metabolically coupled binary heterotroph-photoautotroph system.

Growth of heterotrophic bacterium Bacillus subtilis was metabolically coupled with the photosynthetic activity of an astaxanthin-producing alga Haematococcus pluvialis for conversion of starch-containing waste stream into carotenoid-enriched biomass. The H. pluvialis accounted for 63% of the produced co-culture biomass of 2.2 g/L. Importantly, the binary system requires neither exogenous supply of gaseous substrates nor application of energy-intensive mass transfer technologies due to in-situ exchange in CO2 and O2. The maximum reduction in COD, total nitrogen and phosphorus reached 65%, 55% and 30%, respectively. Conducted techno-economic assessment suggested that the astaxanthin-rich biomass may potentially offset the costs of waste treatment, and, with specific productivity enhancements (induction of astaxanthin to 2% and increase H. pluvialis fraction to 80%), provide and additional revenue stream. The outcome of this study demonstrates a successful proof-of-principle for conversion of waste carbon and nutrients into value-added products through metabolic coupling of heterotrophic and phototrophic metabolisms.

Phytoremediation of agriculture runoff by filamentous algae poly-culture for biomethane production, and nutrient recovery for secondary cultivation of lipid generating microalgae.

An integrated system was implemented for water phytoremediation and biofuel production through sequential cultivation of filamentous algae followed by cultivation of lipid-producing microalgae Chlorella sorokiniana. Natural poly-culture of filamentous algae was grown in agricultural stormwater using the Algal Turf Scrubber®, harvested and subjected for lipid extraction and/or methane production using anaerobic digestion (AD). While filamentous algae lipid content was too low for feasible biodiesel production (<2%), both whole biomass and lipid-extracted algal residues (LEA) yielded ∼0.2LmethanepergVS at loading rates up to 5gVS/L-day. Importantly, essential macro-nutrients and trace elements captured from stormwater were released into the AD effluent as soluble nutrients and were successfully tested as fertilizer replacement for cultivation of lipid-accumulating C. sorokiniana in a subsequent stage. Accordingly, filamentous algae poly-culture was exploited for waste nutrient capturing and biofuel feedstock generation. These nutrients were recovered and reused as a concentrated supplement for potentially high-value microalgae.

Bioprospecting of microalgae for integrated biomass production and phytoremediation of unsterilized wastewater and anaerobic digestion centrate.

Eighteen microalgae, including two local isolates, were evaluated for their ability to grow and remove nutrients from unsterilized primary or secondary wastewater effluents as well as wastewater supplemented with nutrient-rich anaerobic digester centrate (ADC). Most of the tested species except several phylogenetically clustered Chlorella sorokiniana including local isolates and Scenedesmus strains were unable to grow efficiently. This may reflect the presence of certain genetic traits important for robust growth in the unsterilized wastewater. The maximum algal-specific growth rates and biomass density obtained in these bacterial-contaminated cultures were in the range of 0.8-1 day(-1) and 250-350 mg L(-1), respectively. ADC supplementation was especially helpful to biologically treated secondary effluent with its lower initial macronutrient and micronutrient content. As a result of algal growth, total nitrogen and orthophosphate levels were reduced by as much as 90 and 70 %, respectively. Biological assimilation was estimated to be the main mechanism of nitrogen removal in primary and secondary effluents with ammonia volatilization and bacterial nitrification-denitrification contributing for cultures supplemented with ADC. Assimilation by algae served as the principal mechanism of orthophosphate remediation in secondary wastewater cultures, while chemical precipitation appeared also to be important for orthophosphate removal in primary wastewater. Overall, cultivation of microalgae in primary and primary + 5 % ADC may be more favorable from an economical and sustainability perspective due to elimination of the costly and energy-intensive biological treatment step. These findings demonstrate that unsterilized wastewater and ADC can serve as critical nutrient sources for biomass generation and that robust microalgae can be potent players in wastewater phytoremediation.

Anaerobic digestion of lipid-extracted Auxenochlorella protothecoides biomass for methane generation and nutrient recovery.

This study evaluated methane production and nutrient recovery from industrially produced, lipid extracted algal biomass (LEA) of Auxenochlorella protothecoides using semi-continuous anaerobic digestion (AD) at different organic loading rates (OLRs) and hydraulic retention times (HRTs). It was shown, that AD can improve biofuel production efficiency and sustainability, especially for scaled processes, through up to 30% increase in energy generation (up to 0.25 L of methane per g of LEA volatile solids) and partial nutrient recovery and recycling. The nutrient recycling with the AD effluent may reduce the cost of the supplied fertilizers by up to 45%. However, methane production was limited to nearly 50% of theoretical maxima potentially due to biomass recalcitrance and inhibition effects from the residual solvent in the LEA. Therefore, further AD optimization is required to maximize methane yield and nutrient recovery as well as investigation and elimination of inhibition from solvent residues.

Mineral and non-carbon nutrient utilization and recovery during sequential phototrophic-heterotrophic growth of lipid-rich algae.

A critical factor in implementing microalgal biofuels for mass production is the nutrient requirements. The current study investigated the fate of macro- and micronutrients and their availability in a sequential phototrophic-heterotrophic production process for the lipid rich microalga Auxenochlorella protothecoides. More than 99 % (by weight) of overall process nutrients were supplied during the initial photoautotrophic stage reflecting its significantly larger volume. Under photoautotrophic growth conditions only 9-35 % of supplied Mn, S, Fe, N, Mg, and Cu and less than 5 % of P, Mo, Co, B, Zn, and Ca were consumed by the algae. The rest of these nutrients remain in the spent growth media during the culture concentration-down from an 800 L phototrophic pond to a 5 L heterotrophic fermenter. In contrast, Zn, Mo, Mn, Mg, Ca, and N were exhausted (90-99 % removal) during the first 25 h of the heterotrophic growth stage. The depletion of these key nutrients may have ultimately limited the final biomass density and/or lipid productivity achieved. Approximately 10-20 % of the total supplied S, Mn, Fe, N, and Cu and 5 % of Ca and Zn were assimilated into algal biomass. Several elements including N, P, Mn, B, Cu, Ca, Mg, S, and Fe were released back into the liquid phase by anaerobic digestion (AD) of the residual biomass after lipid extraction. The nutrients recovered from the AD effluent and remaining in the spent medium should be recycled or their initial concentration to the phototrophic stage decreased to enhance process economics and sustainability for future commercialization of algal-derived biofuels.

The effects of alternative pretreatment strategies on anaerobic digestion and methane production from different algal strains.

The effect of various pretreatment strategies on methane yields following anaerobic digestion (AD) of five different microalgal strains was investigated. Pavlova_cf sp., Tetraselmis sp. and Thalassiosira weissflogii exhibited substantial methane yields of 0.4-0.5L/g volatile solids (VS) without pretreatment, providing up to 75-80% of theoretical values. In contrast, methane yields from Chlorella sp. and Nannochloropsis sp. were around 0.35L/g VS, or 55-60% of the theoretical values, respectively. Alkali treatment was not effective and thermal pretreatment only enhanced Nannochloropsis methane yields. Thermochemical pretreatment had the strongest impact on biomass solubilization with methane yields increasing by 30% and 40% for Chlorella and Nannochloropsis, respectively. The lipid content had a strong beneficial impact on the theoretical and observed methane yields as compared to protein and carbohydrate content. Other features such as cell-wall composition are also likely to be important factors dictating algal biodegradability and methane yields addressed in part by thermochemical pretreatment.