Genome-scale modeling of light-driven reductant partitioning and carbon fluxes in diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142.

Genome-scale metabolic models have proven useful for answering fundamental questions about metabolic capabilities of a variety of microorganisms, as well as informing their metabolic engineering. However, only a few models are available for oxygenic photosynthetic microorganisms, particularly in cyanobacteria in which photosynthetic and respiratory electron transport chains (ETC) share components. We addressed the complexity of cyanobacterial ETC by developing a genome-scale model for the diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142. The resulting metabolic reconstruction, iCce806, consists of 806 genes associated with 667 metabolic reactions and includes a detailed representation of the ETC and a biomass equation based on experimental measurements. Both computational and experimental approaches were used to investigate light-driven metabolism in Cyanothece sp. ATCC 51142, with a particular focus on reductant production and partitioning within the ETC. The simulation results suggest that growth and metabolic flux distributions are substantially impacted by the relative amounts of light going into the individual photosystems. When growth is limited by the flux through photosystem I, terminal respiratory oxidases are predicted to be an important mechanism for removing excess reductant. Similarly, under photosystem II flux limitation, excess electron carriers must be removed via cyclic electron transport. Furthermore, in silico calculations were in good quantitative agreement with the measured growth rates whereas predictions of reaction usage were qualitatively consistent with protein and mRNA expression data, which we used to further improve the resolution of intracellular flux values.

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.

Conversion of L-lactate into n-caproate by a continuously fed reactor microbiome.

Conversion of lactate to n-caproate had been described for the type strain Megasphaera elsdenii in batch systems. Recently, investigators have also described production of n-caproate from endogenous or exogenous lactate with batch-fed reactor microbiome systems. However, no reports exist of lactate to n-caproate conversion within a continuously fed bioreactor. Since continuously fed systems are advantageous for biotechnology production platforms, our objective was to develop such a system. Here, we demonstrated continuous lactate to n-caproate conversion for more than 165 days. The volumetric n-caproate production rate (productivity) was improved when we decreased the operating pH from 5.5 to 5.0, and was again improved when we utilized in-line product recovery via pertraction (membrane-based liquid-liquid extraction). We observed a maximum n-caproate productivity of 6.9 g COD/L-d for a period of 17 days at an L-lactate loading rate of 9.1 g COD/L-d, representing the highest sustained lactate to n-caproate conversion rate ever reported. We had to manage two competing lactate conversion pathways: 1) the reverse ß-oxidation pathway to n-caproate; and 2) the acrylate pathway to propionate. We found that maintaining a low residual lactate concentration in the bioreactor broth was necessary to direct lactate conversion towards n-caproate instead of propionate. These findings provide a foundation for the development of new resource recovery processes to produce higher-value liquid products (e.g., n-caproate) from carbon-rich wastewaters containing lactate or lactate precursors (e.g., carbohydrates).

Waste Conversion into n-Caprylate and n-Caproate: Resource Recovery from Wine Lees Using Anaerobic Reactor Microbiomes and In-line Extraction.

To convert wastes into sustainable liquid fuels and chemicals, new resource recovery technologies are required. Chain elongation is a carboxylate-platform bioprocess that converts short-chain carboxylates (SCCs) (e.g., acetate [C2] and n-butyrate [C4]) into medium-chain carboxylates (MCCs) (e.g., n-caprylate [C8] and n-caproate [C6]) with hydrogen gas as a side product. Ethanol or another electron donor (e.g., lactate, carbohydrate) is required. Competitive MCC productivities, yields (product vs. substrate fed), and specificities (product vs. all products) were only achieved previously from an organic waste material when exogenous ethanol had been added. Here, we converted a real organic waste, which inherently contains ethanol, into MCCs with n-caprylate as the target product. We used wine lees, which consisted primarily of settled yeast cells and ethanol from wine fermentation, and produced MCCs with a reactor microbiome. We operated the bioreactor at a pH of 5.2 and with continuous in-line extraction and achieved a MCC productivity of 3.9 g COD/L-d at an organic loading rate of 5.8 g COD/L-d, resulting in a promising MCC yield of 67% and specificities of 36% for each n-caprylate and n-caproate (72% for both). Compared to all other studies that used complex organic substrates, we achieved the highest n-caprylate-to-ncaproate product ratio of 1.0 (COD basis), because we used increased broth-recycle rates through the forward membrane contactor, which improved in-line extraction rates. Increased recycle rates also allowed us to achieve the highest reported MCC production flux per membrane surface area thus far (20.1 g COD/m2-d). Through microbial community analyses, we determined that an operational taxonomic unit (OTU) for Bacteroides spp. was dominant and was positively correlated with increased MCC productivities. Our data also suggested that the microbiome may have been shaped for improved MCC production by the high broth-recycle rates. Comparable abiotic studies suggest that further increases in the broth-recycle rates could improve the overall mass transfer coefficient and its corresponding MCC production flux by almost 30 times beyond the maximum that we achieved. With improved in-line extraction, the chain-elongation biotechnology production platform offers new opportunities for resource recovery and sustainable production of liquid fuels and chemicals.

Lauric Acid Production in a Glycogen-Less Strain of Synechococcus sp. PCC 7002.

The cyanobacterium Synechococcus sp. Pasteur culture collection 7002 was genetically engineered to synthesize biofuel-compatible medium-chain fatty acids (FAs) during photoautotrophic growth. Expression of a heterologous lauroyl-acyl carrier protein (C12:0-ACP) thioesterase with concurrent deletion of the endogenous putative acyl-ACP synthetase led to secretion of transesterifiable C12:0 FA in CO2-supplemented batch cultures. When grown at steady state over a range of light intensities in a light-emitting diode turbidostat photobioreactor, the C12-secreting mutant exhibited a modest reduction in growth rate and increased O2 evolution relative to the wild-type (WT). Inhibition of (i) glycogen synthesis by deletion of the glgC-encoded ADP-glucose pyrophosphorylase (AGPase) and (ii) protein synthesis by nitrogen deprivation were investigated as potential mechanisms for metabolite redistribution to increase FA synthesis. Deletion of AGPase led to a 10-fold decrease in reducing carbohydrates and secretion of organic acids during nitrogen deprivation consistent with an energy spilling phenotype. When the carbohydrate-deficient background (ΔglgC) was modified for C12 secretion, no increase in C12 was achieved during nutrient replete growth, and no C12 was recovered from any strain upon nitrogen deprivation under the conditions used. At steady state, the growth rate of the ΔglgC strain saturated at a lower light intensity than the WT, but O2 evolution was not compromised and became increasingly decoupled from growth rate with rising irradiance. Photophysiological properties of the ΔglgC strain suggest energy dissipation from photosystem II and reconfiguration of electron flow at the level of the plastoquinone pool.

Effect of mono- and dichromatic light quality on growth rates and photosynthetic performance of Synechococcus sp. PCC 7002.

Synechococcus sp. PCC 7002 was grown to steady state in optically thin turbidostat cultures under conditions for which light quantity and quality was systematically varied by modulating the output of narrow-band LEDs. Cells were provided photons absorbed primarily by chlorophyll (680 nm) or phycocyanin (630 nm) as the organism was subjected to four distinct mono- and dichromatic regimes. During cultivation with dichromatic light, growth rates were generally proportional to the total incident irradiance at values <275 µmol photons m(-2) · s(-1) and were not affected by the ratio of 630:680 nm wavelengths. Notably, under monochromatic light conditions, cultures exhibited similar growth rates only when they were irradiated with 630 nm light; cultures irradiated with only 680 nm light grew at rates that were 60-70% of those under other light quality regimes at equivalent irradiances. The functionality of photosystem II and associated processes such as maximum rate of photosynthetic electron transport, rate of cyclic electron flow, and rate of dark respiration generally increased as a function of growth rate. Nonetheless, some of the photophysiological parameters measured here displayed distinct patterns with respect to growth rate of cultures adapted to a single wavelength including phycobiliprotein content, which increased under severely light-limited growth conditions. Additionally, the ratio of photosystem II to photosystem I increased ~40% over the range of growth rates, although cells grown with 680 nm light only had the highest ratios. These results suggest the presence of effective mechanisms which allow acclimation of Synechococcus sp. PCC 7002 acclimation to different irradiance conditions.

Inference of interactions in cyanobacterial-heterotrophic co-cultures via transcriptome sequencing.

We used deep sequencing technology to identify transcriptional adaptation of the euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 and the marine facultative aerobe Shewanella putrefaciens W3-18-1 to growth in a co-culture and infer the effect of carbon flux distributions on photoautotroph-heterotroph interactions. The overall transcriptome response of both organisms to co-cultivation was shaped by their respective physiologies and growth constraints. Carbon limitation resulted in the expansion of metabolic capacities, which was manifested through the transcriptional upregulation of transport and catabolic pathways. Although growth coupling occurred via lactate oxidation or secretion of photosynthetically fixed carbon, there was evidence of specific metabolic interactions between the two organisms. These hypothesized interactions were inferred from the excretion of specific amino acids (for example, alanine and methionine) by the cyanobacterium, which correlated with the downregulation of the corresponding biosynthetic machinery in Shewanella W3-18-1. In addition, the broad and consistent decrease of mRNA levels for many Fe-regulated Synechococcus 7002 genes during co-cultivation may indicate increased Fe availability as well as more facile and energy-efficient mechanisms for Fe acquisition by the cyanobacterium. Furthermore, evidence pointed at potentially novel interactions between oxygenic photoautotrophs and heterotrophs related to the oxidative stress response as transcriptional patterns suggested that Synechococcus 7002 rather than Shewanella W3-18-1 provided scavenging functions for reactive oxygen species under co-culture conditions. This study provides an initial insight into the complexity of photoautotrophic-heterotrophic interactions and brings new perspectives of their role in the robustness and stability of the association.

Computational evaluation of Synechococcus sp. PCC 7002 metabolism for chemical production.

Cyanobacteria are ideal metabolic engineering platforms for carbon-neutral biotechnology because they directly convert CO2 to a range of valuable products. In this study, we present a computational assessment of biochemical production in Synechococcus sp. PCC 7002 (Synechococcus 7002), a fast growing cyanobacterium whose genome has been sequenced, and for which genetic modification methods have been developed. We evaluated the maximum theoretical yields (mol product per mol CO2 or mol photon) of producing various chemicals under photoautotrophic and dark conditions using a genome-scale metabolic model of Synechococcus 7002. We found that the yields were lower under dark conditions, compared to photoautotrophic conditions, due to the limited amount of energy and reductant generated from glycogen. We also examined the effects of photon and CO2 limitations on chemical production under photoautotrophic conditions. In addition, using various computational methods such as minimization of metabolic adjustment (MOMA), relative metabolic change (RELATCH), and OptORF, we identified gene-knockout mutants that are predicted to improve chemical production under photoautotrophic and/or dark anoxic conditions. These computational results are useful for metabolic engineering of cyanobacteria to synthesize value-added products.

Feedback-controlled LED photobioreactor for photophysiological studies of cyanobacteria.

A custom photobioreactor was designed to enable automatic light adjustments using computerized feedback control. The system consisted of a 7.5-L cylindrical vessel and an aluminum enclosure housing quantum sensors and light-emitting diode arrays, which provide 630 or 680 nm light to preferentially excite the major cyanobacterial pigments, phycocyanin and/or chlorophyll a, respectively. Custom-developed software rapidly measures light transmission and subsequently adjusts the irradiance to maintain a defined light profile to compensate for culture dynamics, biomass accumulation, and pigment adaptations during physiological transitions, thus ensuring appropriate illumination across batch and continuous growth modes. In addition to chemostat cultivation, the photobioreactor may also operate as a turbidostat, continuously adjusting the media dilution to achieve maximal growth at a fixed culture density. The cultivation system doubles as an analytical device, using real-time monitoring to avoid sampling bias (e.g., in-situ light-saturation response), determine conditions for optimal growth, and observe perturbation responses at high time-resolution.

Sustained H(2) production driven by photosynthetic water splitting in a unicellular cyanobacterium.

UNLABELLED: The relationship between dinitrogenase-driven H(2) production and oxygenic photosynthesis was investigated in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142, using a novel custom-built photobioreactor equipped with advanced process control. Continuously illuminated nitrogen-deprived cells evolved H(2) at rates up to 400 µmol ⋅ mg Chl(-1) ⋅ h(-1) in parallel with uninterrupted photosynthetic O(2) production. Notably, sustained coproduction of H(2) and O(2) occurred over 100 h in the presence of CO(2), with both gases displaying inverse oscillations which eventually dampened toward stable rates of 125 and 90 µmol ⋅ mg Chl(-1) ⋅ h(-1), respectively. Oscillations were not observed when CO(2) was omitted, and instead H(2) and O(2) evolution rates were positively correlated. The sustainability of the process was further supported by stable chlorophyll content, maintenance of baseline protein and carbohydrate levels, and an enhanced capacity for linear electron transport as measured by chlorophyll fluorescence throughout the experiment. In situ light saturation analyses of H(2) production displayed a strong dose dependence and lack of O(2) inhibition. Inactivation of photosystem II had substantial long-term effects but did not affect short-term H(2) production, indicating that the process is also supported by photosystem I activity and oxidation of endogenous glycogen. However, mass balance calculations suggest that carbohydrate consumption in the light may, at best, account for no more than 50% of the reductant required for the corresponding H(2) production over that period. Collectively, our results demonstrate that uninterrupted H(2) production in unicellular cyanobacteria can be fueled by water photolysis without the detrimental effects of O(2) and have important implications for sustainable production of biofuels. IMPORTANCE: The study provides an important insight into the photophysiology of light-driven H(2) production by the nitrogen-fixing cyanobacterium Cyanothece sp. strain ATCC 51142. This work is also of significance for biotechnology, supporting the feasibility of "direct biophotolysis." The sustainability of the process, highlighted by prolonged gas evolution with no clear sign of significant decay or apparent photodamage, provides a foundation for the future development of an effective, renewable, and economically efficient bio-H(2) production process.