A gene regulatory network for antenna size control in carbon dioxide-deprived Chlamydomonas reinhardtii cells.

In green microalgae, prolonged exposure to inorganic carbon depletion requires long-term acclimation responses, involving modulated gene expression and the adjustment of photosynthetic activity to the prevailing supply of carbon dioxide. Here, we describe a microalgal regulatory cycle that adjusts the light-harvesting capacity at photosystem II (PSII) to the prevailing supply of carbon dioxide in Chlamydomonas (Chlamydomonas reinhardtii). It engages low carbon dioxide response factor (LCRF), a member of the squamosa promoter-binding protein (SBP) family of transcription factors, and the previously characterized cytosolic translation repressor nucleic acid-binding protein 1 (NAB1). LCRF combines a DNA-binding SBP domain with a conserved domain for protein-protein interaction. LCRF transcription is rapidly induced by carbon dioxide depletion. LCRF activates NAB1 transcription by specifically binding to tetranucleotide motifs present in its promoter. Accumulation of the NAB1 protein enhances translational repression of its prime target mRNA, encoding the PSII-associated major light-harvesting protein LHCBM6. The resulting truncation of the PSII antenna size helps maintaining a low excitation during carbon dioxide limitation. Analyses of low carbon dioxide acclimation in nuclear insertion mutants devoid of a functional LCRF gene confirm the essentiality of this novel transcription factor for the regulatory circuit.

Light-Harvesting Complex Protein LHCBM9 Is Critical for Photosystem II Activity and Hydrogen Production in Chlamydomonas reinhardtii.

Photosynthetic organisms developed multiple strategies for balancing light-harvesting versus intracellular energy utilization to survive ever-changing environmental conditions. The light-harvesting complex (LHC) protein family is of paramount importance for this function and can form light-harvesting pigment protein complexes. In this work, we describe detailed analyses of the photosystem II (PSII) LHC protein LHCBM9 of the microalga Chlamydomonas reinhardtii in terms of expression kinetics, localization, and function. In contrast to most LHC members described before, LHCBM9 expression was determined to be very low during standard cell cultivation but strongly increased as a response to specific stress conditions, e.g., when nutrient availability was limited. LHCBM9 was localized as part of PSII supercomplexes but was not found in association with photosystem I complexes. Knockdown cell lines with 50 to 70% reduced amounts of LHCBM9 showed reduced photosynthetic activity upon illumination and severe perturbation of hydrogen production activity. Functional analysis, performed on isolated PSII supercomplexes and recombinant LHCBM9 proteins, demonstrated that presence of LHCBM9 resulted in faster chlorophyll fluorescence decay and reduced production of singlet oxygen, indicating upgraded photoprotection. We conclude that LHCBM9 has a special role within the family of LHCII proteins and serves an important protective function during stress conditions by promoting efficient light energy dissipation and stabilizing PSII supercomplexes.

Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production.

BACKGROUND: Microalgae are gaining importance as sustainable production hosts in the fields of biotechnology and bioenergy. A robust biomass accumulating strain of the genus Monoraphidium (SAG 48.87) was investigated in this work as a potential feedstock for biofuel production. The genome was sequenced, annotated, and key enzymes for triacylglycerol formation were elucidated. RESULTS: Monoraphidium neglectum was identified as an oleaginous species with favourable growth characteristics as well as a high potential for crude oil production, based on neutral lipid contents of approximately 21% (dry weight) under nitrogen starvation, composed of predominantly C18:1 and C16:0 fatty acids. Further characterization revealed growth in a relatively wide pH range and salt concentrations of up to 1.0% NaCl, in which the cells exhibited larger structures. This first full genome sequencing of a member of the Selenastraceae revealed a diploid, approximately 68 Mbp genome with a G + C content of 64.7%. The circular chloroplast genome was assembled to a 135,362 bp single contig, containing 67 protein-coding genes. The assembly of the mitochondrial genome resulted in two contigs with an approximate total size of 94 kb, the largest known mitochondrial genome within algae. 16,761 protein-coding genes were assigned to the nuclear genome. Comparison of gene sets with respect to functional categories revealed a higher gene number assigned to the category "carbohydrate metabolic process" and in "fatty acid biosynthetic process" in M. neglectum when compared to Chlamydomonas reinhardtii and Nannochloropsis gaditana, indicating a higher metabolic diversity for applications in carbohydrate conversions of biotechnological relevance. CONCLUSIONS: The genome of M. neglectum, as well as the metabolic reconstruction of crucial lipid pathways, provides new insights into the diversity of the lipid metabolism in microalgae. The results of this work provide a platform to encourage the development of this strain for biotechnological applications and production concepts.

Microbial Diversity and Community Structure of Wastewater-Driven Microalgal Biofilms.

Dwindling water sources increase the need for efficient wastewater treatment. Solar-driven algal turf scrubber (ATS) system may remediate wastewater by supporting the development and growth of periphytic microbiomes that function and interact in a highly dynamic manner through symbiotic interactions. Using ITS and 16S rRNA gene amplicon sequencing, we profiled the microbial communities of four microbial biofilms from ATS systems operated with municipal wastewater (mWW), diluted cattle and pig manure (CattleM and PigM), and biogas plant effluent supernatant (BGE) in comparison to the initial inocula and the respective wastewater substrates. The wastewater-driven biofilms differed significantly in their biodiversity and structure, exhibiting an inocula-independent but substrate-dependent establishment of the microbial communities. The prokaryotic communities were comparable among themselves and with other microbiomes of aquatic environments and were dominated by metabolically flexible prokaryotes such as nitrifiers, polyphosphate-accumulating and algicide-producing microorganisms, and anoxygenic photoautotrophs. Striking differences occurred in eukaryotic communities: While the mWW biofilm was characterized by high biodiversity and many filamentous (benthic) microalgae, the agricultural wastewater-fed biofilms consisted of less diverse communities with few benthic taxa mainly inhabited by unicellular chlorophytes and saprophytes/parasites. This study advances our understanding of the microbiome structure and function within the ATS-based wastewater treatment process.

Phytoplankton consortia as a blueprint for mutually beneficial eukaryote-bacteria ecosystems based on the biocoenosis of Botryococcus consortia.

Bacteria occupy all major ecosystems and maintain an intensive relationship to the eukaryotes, developing together into complex biomes (i.e., phycosphere and rhizosphere). Interactions between eukaryotes and bacteria range from cooperative to competitive, with the associated microorganisms affecting their host`s development, growth and health. Since the advent of non-culture dependent analytical techniques such as metagenome sequencing, consortia have been described at the phylogenetic level but rarely functionally. Multifaceted analysis of the microbial consortium of the ancient phytoplankton Botryococcus as an attractive model food web revealed that its all abundant bacterial members belong to a niche of biotin auxotrophs, essentially depending on the microalga. In addition, hydrocarbonoclastic bacteria without vitamin auxotrophies seem adversely to affect the algal cell morphology. Synthetic rearrangement of a minimal community consisting of an alga, a mutualistic and a parasitic bacteria underpins the model of a eukaryote that maintains its own mutualistic microbial community to control its surrounding biosphere. This model of coexistence, potentially useful for defense against invaders by a eukaryotic host could represent ecologically relevant interactions that cross species boundaries. Metabolic and system reconstruction is an opportunity to unravel the relationships within the consortia and provide a blueprint for the construction of mutually beneficial synthetic ecosystems.

Wastewater-borne microalga Chlamydomonas sp.: A robust chassis for efficient biomass and biomethane production applying low-N cultivation strategy.

Biogas/biomethane generation from microalgae biomass via anaerobic fermentation is increasingly gaining attention as CO2-neutral energy source. Intensive research has shown, however, that microalgae represent a rather challenging substrate for anaerobic digestion (AD) due to their high cell wall recalcitrance and unfavourable protein content. Previously, the utilization of nitrogen-limited (low-N) microalgal biomass for continuous AD-processes was demonstrated (as proof-of-concept) with remarkable biomethane productivity. The present study shows the efficient portability of the low-N cultivation/fermentation strategy on a robust, wastewater-borne microalga isolate that tolerates high temperature and light conditions and can perfectly cope with microbial contaminations. Continuous long-term anaerobic digestion was characterized by stable and efficient specific biogas and biomethane productivity (765 ± 20 and 478 ± 15 mLNg-1 volatile solids (VS) d-1, respectively), equivalent to volumetric methane productivity of 1912 mLN L-1d-1. The present work underlines the applicability of low-N-biomass of wastewater-borne, robust microalgae as mono-substrate for highly efficient continuous methane generation.

Type II flavoprotein monooxygenase PsFMO_A from the bacterium Pimelobacter sp. Bb-B catalyzes enantioselective Baeyer-Villiger oxidations with a relaxed cofactor specificity.

Microbial consortia, which degrade branched, long-chain hydrocarbons, can be regarded as a promising source of novel enzymes for the stereo- and regio-selective oxyfunctionalization of hydrocarbons. The hydrocarbon-degrading bacterium Pimelobacter sp. Bb-B was isolated from the consortium associated with the colonial hydrocarbon-excreting microalga Botryococcus braunii. Three new type II flavoprotein monooxygenases (type II FMOs) from this bacterium have been made available in recombinant form through cloning and overexpression in an E. coli host organism. These enzymes (PsFMO_A-C) were characterized in terms of their capability of catalyzing Baeyer-Villiger oxidations with distinct substrates. The highest activity was detected when utilizing camphor and bicyclo[3.2.0]hept-2-en-6-one as substrate in combination with PsFMO_A as the most promising enzyme. Furthermore, synthetic biotransformations with 5 mM of the substrate bicyclo[3.2.0]hept-2-en-6-one, formate and formate dehydrogenase for in situ-cofactor recycling were conducted with this enzyme, leading to a substrate consumption of 85% after 66 h and excellent enantioselectivity of 99% ee for the (1R,5S)-enantiomer. Additionally, an alternative in situ-cofactor recycling based on the use of microalgae for in situ-production of formate from carbon dioxide, water and light together with a formate dehydrogenase was combined successfully with the enzyme PsFMO_A, leading to a substrate consumption of 94% and an enantioselectivity of >99% ee for the so-called "normal lactone"-enantiomer with the absolute configuration 1R,5S.

Metabolic survey of Botryococcus braunii: Impact of the physiological state on product formation.

The microalga Botryococcus braunii is widely regarded as a potential renewable and sustainable source for industrial applications because of its capability to produce large amounts of metabolically expensive (exo-) polysaccharides and lipids, notably hydrocarbons. A comprehensive and systematic metabolic characterization of the Botryococcus braunii race A strain CCAP 807/2 was conducted within the present study, including the detailed analysis of growth-associated and physiological parameters. In addition, the intracellular metabolome was profiled for the first time and showed growth- and product-specific fluctuations in response to the different availability of medium resources during the cultivation course. Among the identified metabolites, a constant expression of raffinose was observed for the first time under standard conditions, which has until now only been described for higher plants. Overall, the multilayered analysis during the cultivation of strain CCAP 807/2 allowed the differentiation of four distinct physiological growth phases and revealed differences in the production profiles and content of liquid hydrocarbons and carbohydrates with up to 84% of organic dry weight (oDW). In the process, an enhanced production of carbohydrates with up to 63% of oDW (1.36±0.03 g L-1) could be observed during the late linear growth phase, whereas the highest accumulation of extracellular hydrocarbons with up to 24% of oDW (0.66±0.12 g L-1) occurred mainly during the stationary growth phase. Altogether, the knowledge obtained is potentially useful for the general understanding of the overall physiology of Botryococcus braunii and provide important insights into the growth behavior and product formation of this microalga, and is thus relevant for large scale biofuel production and industrial applications.

Highly efficient methane generation from untreated microalgae biomass.

BACKGROUND: The fact that microalgae perform very efficiently photosynthetic conversion of sunlight into chemical energy has moved them into the focus of regenerative fuel research. Especially, biogas generation via anaerobic digestion is economically attractive due to the comparably simple apparative process technology and the theoretical possibility of converting the entire algal biomass to biogas/methane. In the last 60 years, intensive research on biogas production from microalgae biomass has revealed the microalgae as a rather challenging substrate for anaerobic digestion due to its high cell wall recalcitrance and unfavorable protein content, which requires additional pretreatment and co-fermentation strategies for sufficient fermentation. However, sustainable fuel generation requires the avoidance of cost/energy intensive biomass pretreatments to achieve positive net-energy process balance. RESULTS: Cultivation of microalgae in replete and limited nitrogen culture media conditions has led to the formation of protein-rich and low protein biomass, respectively, with the last being especially optimal for continuous fermentation. Anaerobic digestion of nitrogen limited biomass (low-N BM) was characterized by a stable process with low levels of inhibitory substances and resulted in extraordinary high biogas, and subsequently methane productivity [750 ± 15 and 462 ± 9 mLN g-1 volatile solids (VS) day-1, respectively], thus corresponding to biomass-to-methane energy conversion efficiency of up to 84%. The microbial community structure within this highly efficient digester revealed a clear predominance of the phyla Bacteroidetes and the family Methanosaetaceae among the Bacteria and Archaea, respectively. The fermentation of replete nitrogen biomass (replete-N BM), on the contrary, was demonstrated to be less productive (131 ± 33 mLN CH4 g-1VS day-1) and failed completely due to acidosis, caused through high ammonia/ammonium concentrations. The organization of the microbial community of the failed (replete-N) digester differed greatly compared to the stable low-N digester, presenting a clear shift to the phyla Firmicutes and Thermotogae, and the archaeal population shifted from acetoclastic to hydrogenotrophic methanogenesis. CONCLUSIONS: The present study underlines the importance of cultivation conditions and shows the practicability of microalgae biomass usage as mono-substrate for highly efficient continuous fermentation to methane without any pretreatment with almost maximum practically achievable energy conversion efficiency (biomass to methane).Graphical abstractGrowth condition dependence of anaerobic conversion efficiency of microalgae biomass to methane.

Efficiency and biotechnological aspects of biogas production from microalgal substrates.

Photosynthetic organisms like plants and algae can harvest, convert, and store solar energy and thus represent readily available sources for renewable biofuels production on a domestic or industrial scale. Anaerobic digestion (AD) of the organic biomass yields biogas, containing methane and carbon dioxide as major constituents. Combustion of the biogas or purification of the energy-rich methane fraction can be applied to provide electricity or fuel. AD procedures have been applied for several decades with organic waste, animal products, or higher plants and more recently, utilization of photosynthetic algae as substrates have gained considerable research interest. To provide an overview of recent research efforts made to characterize the AD process of microalgal biomass, we present extended summaries of experimentally determined biochemical methane potentials (BMP), biomass pretreatment options and digestion strategies in this article. We conclude that cultivation options, biomass composition and time of harvesting, application of biomass pretreatment strategies, and parameters of the digestion process are all important factors, which can significantly affect the AD process efficiency. The transition from batch to continuous microalgal biomass digestion trials, accompanied by state-of-the-art analytical techniques, is now in demand to refine the assessments of the overall process feasibility.

Complete Chloroplast and Mitochondrial Genome Sequences of the Hydrocarbon Oil-Producing Green Microalga Botryococcus braunii Race B (Showa).

The green alga Botryococcus braunii is capable of the production and excretion of high quantities of long-chain hydrocarbons and exopolysaccharides. In this study, we present the complete plastid and mitochondrial genomes of the hydrocarbon-producing microalga Botryococcus braunii race B (Showa), with a total length of 156,498 and 129,356 bp, respectively.

A novel one-stage cultivation/fermentation strategy for improved biogas production with microalgal biomass.

The use of alga biomass for biogas generation has been studied for over fifty years but until today, several distinct features, like inefficient degradation and low C/N ratios, limit the applicability of algal biomass for biogas production in larger scale. In this work we investigated a novel, one-stage combined cultivation/fermentation strategy including inherently progressing nitrogen starvation conditions to generate improved microalgal biomass substrates. For this strategy, comparable low amounts of nitrogen fertilizers were applied during cultivation and no additional enzymatic, chemical or physical pretreatments had to be performed. The results of this study demonstrate that progressing nitrogen limitation leads to continuously increasing C/N ratios of the biomass up to levels of 24-26 for all three tested alga strains (Chlamydomonas reinhardtii, Parachlorella kessleri and Scenedesmus obliquus). Importantly, the degradation efficiency of the algal cells increased with progressing starvation, leading to strain-specific cell disintegration efficiencies of 35%-100% during the fermentation process. Nitrogen limitation treatment resulted in a 65% increase of biogas yields for C. reinhardtii biomass (max. 698±23mL biogas g(-1) VS) when compared to replete conditions. For P. kessleri and S. obliquus, yields increased by 94% and 106% (max. 706±39mL and 586±36mL biogas g(-1) VS, respectively). From these results we conclude that this novel one-stage cultivation strategy with inherent nitrogen limitation can be used as a pretreatment for microalgal biomass generation, in order to produce accessible substrates with optimized C/N ratios for the subsequent anaerobic fermentation process, thus increasing methane production and avoiding the risk of ammonia inhibition effects within the fermenter.

Integration of carbon assimilation modes with photosynthetic light capture in the green alga Chlamydomonas reinhardtii.

The unicellular green alga Chlamydomonas reinhardtii is capable of using organic and inorganic carbon sources simultaneously, which requires the adjustment of photosynthetic activity to the prevailing mode of carbon assimilation. We obtained novel insights into the regulation of light-harvesting at photosystem II (PSII) following altered carbon source availability. In C. reinhardtii, synthesis of PSII-associated light-harvesting proteins (LHCBMs) is controlled by the cytosolic RNA-binding protein NAB1, which represses translation of particular LHCBM isoform transcripts. This mechanism is fine-tuned via regulation of the nuclear NAB1 promoter, which is activated when linear photosynthetic electron flow is restricted by CO(2)-limitation in a photoheterotrophic context. In the wild-type, accumulation of NAB1 reduces the functional PSII antenna size, thus preventing a harmful overexcited state of PSII, as observed in a NAB1-less mutant. We further demonstrate that translation control as a newly identified long-term response to prolonged CO(2)-limitation replaces LHCII state transitions as a fast response to PSII over-excitation. Intriguingly, activation of the long-term response is perturbed in state transition mutant stt7, suggesting a regulatory link between the long- and short-term response. We depict a regulatory circuit operating on distinct timescales and in different cellular compartments to fine-tune light-harvesting in photoheterotrophic eukaryotes.

Cellulose degradation and assimilation by the unicellular phototrophic eukaryote Chlamydomonas reinhardtii.

Plants convert sunlight to biomass, which is primarily composed of lignocellulose, the most abundant natural biopolymer and a potential feedstock for fuel and chemical production. Cellulose assimilation has so far only been described for heterotrophic organisms that rely on photosynthetically active primary producers of organic compounds. Among phototrophs, the unicellular green microalga Chlamydomonas reinhardtii is widely known as one of the best established model organisms. It occupies many habitats, including aquatic and soil ecosystems. This ubiquity underscores the versatile metabolic properties of this microorganism. Here we present yet another paradigm of adaptation for C. reinhardtii, highlighting its photoheterotrophic ability to utilize cellulose for growth in the absence of other carbon sources. When grown under CO(2)-limiting conditions in the light, secretion of endo-ß-1,4-glucanases by the cell causes digestion of exogenous cellulose, followed by cellobiose uptake and assimilation. Phototrophic microbes like C. reinhardtii may thus serve as biocatalysts for cellulosic biofuel production.