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

Klassen V, Blifernez-Klassen O, Hoekzema Y, Mussgnug JH, Kruse O. A novel one-stage cultivation/fermentation strategy for improved biogas production with microalgal biomass. Journal of Biotechnology. 2015;215:44-51

Highly efficient methane generation from untreated microalgae biomass

Klassen V, Blifernez-Klassen O, Wibberg D, et al. Highly efficient methane generation from untreated microalgae biomass. Biotechnology for Biofuels. 2017;10(1): 186.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)

Complete Chloroplast and Mitochondrial Genome Sequences of the Hydrocarbon Oil-Producing Green MicroalgaBotryococcus brauniiRace B (Showa)

Blifernez-Klassen O, Wibberg D, Winkler A, et al. Complete Chloroplast and Mitochondrial Genome Sequences of the Hydrocarbon Oil-Producing Green MicroalgaBotryococcus brauniiRace B (Showa). Genome Announcements. 2016;4(3): e00524-16.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

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

Bogen C, Al-Dilaimi A, Albersmeier A, et al. Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production. BMC Genomics. 2013;14(1): 926.BACKGROUND: Microalgae are gaining importance as sustainable production hosts in the fields of biotechnology and bioenergy. A robust biomass accumulating strainof the genus Monoraphidium (SAG 48.87) was investigated in this work as apotential 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

Molecular mechanisms behind the adjustment of phototrophic light-harvesting and mixotrophic utilization of cellulosic carbon sources in Chlamydomonas reinhardtii

Blifernez-Klassen O. Molecular mechanisms behind the adjustment of phototrophic light-harvesting and mixotrophic utilization of cellulosic carbon sources in Chlamydomonas reinhardtii. Bielefeld: Universität Bielefeld; 2012.Plants, green algae and cyanobacteria perform photosynthetic conversion of sunlight into chemical energy in a permanently changing natural environment, where the efficient utilization of light and inorganic carbon represent the most critical factors. Photosynthetic organisms have developed different acclimation strategies to adapt changing light conditions and insufficient carbon source supply in order to survive and to assure optimal growth and protection. This thesis provides further insights into the molecular mechanisms of the acclimation response of the green algae Chlamydomonas reinhardtii. An important acclimation mechanism to altering light conditions involves the post-transcriptional regulation of the nuclear-encoded photosystem II (PSII)-associated light-harvesting complex (LHCII) genes via cytosolic translational control mediated through the RNA-binding protein NAB1. In the active state, NAB1 represses the cytosolic translation of LHCII mRNAs by sequestration into translational silent messenger ribonucleoprotein complexes (mRNPs). The overexpression of NAB1 decreases LHCII protein amount whereas NAB1 knockout leads to an increased level of LHCII proteins. Consequently, NAB1 is part of a control system regulating the size and composition of the LHCII complex at the posttranscriptional level. The repressor activity of this specific factor is controlled by two posttranslational modifications: i) by methylation of arginines in the glycine-arginine rich (GAR) motif of the protein, ii) by the thiol status of two C-terminal cysteines. This work provides evidence that arginine methylation represents a slowly reacting modulator, which is required for the maintenance of the repressor activity of NAB1 and is responsive to the availability of light. At the same time, cysteine modification is regarded as the fine-tuning mechanism that dynamically responds to changes in the cytosolic redox-state. Moreover, the observations suggest that the regulation via arginine methylation operates independently from cysteine-based redox control, with its extent strongly depending on the growth conditions. The high methylation state is found under photoautotrophic, and the low methylation state under heterotrophic growth conditions. Photosynthetic performance is also dependent on inorganic carbon (Ci) supply, because the light-harvesting capacity and the utilization of captured energy have to be balanced. The insufficient Ci-availability can be compensated by diverse organic carbon sources, since some phototrophs can assimilate acetate, glucose or other sugars for mixothrophic growth. The green alga C. reinhardtii was so far reported to grow on acetate, but not on hexoses. Intriguingly, in silico analysis of the genome of C. reinhardtii revealed that it contains genes encoding glycoside hydrolases of different families, known to be involved in cellulose- and hemicellulose degradation, even though the cell wall of this alga does not contain cellulose and is solely composed of hydroxyproline-rich glycoproteins. This work characterizes the capability of C. reinhardtii for cellulose degradation as seen by the digestion of cellulosic materials such as carboxymethyl cellulose, Avicel and filter paper. Furthermore, the results of the present work indicate an assimilation of the breakdown products, in particular cellobiose. Cellulose degradation into cellodextrins (cellobiose-cellopentaose) was shown to be performed by extracellular cellulases CrCel9B and CrCel9C that belong to glycoside hydrolase family 9 (GHF9/subgroup E2). These enzymes display homology to cellulases from Metazoa and phylogenetic analyses suggested that they originated from an ancient eukaryotic ancestor. Furthermore, a positive effect on specific growth rates was observable under different growth conditions (high, low and very-low CO2 as well as acetate) after media supplementation with cellulosic material. In conclusion, this work provides advanced insights into the molecular regulation mechanisms of light-acclimation and utilization of an abundant organic carbon source in Chlamydomonas reinhardtii. These new findings could help to achieve higher biomass productivity by improved photosynthetic conversion efficiency and additionally, the use of abundant organic carbon sources as an integral part of new photoheterotrophic cultivation concepts

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

Klassen V, Blifernez-Klassen O, Bax J, Kruse O. Wastewater-borne microalga Chlamydomonas sp.: A robust chassis for efficient biomass and biomethane production applying low-N cultivation strategy. Bioresource Technology. 2020;315: 123825.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

Microbial Diversity and Community Structure of Wastewater-Driven Microalgal Biofilms

Blifernez-Klassen O, Hassa J, Reinecke DL, Busche T, Klassen V, Kruse O. Microbial Diversity and Community Structure of Wastewater-Driven Microalgal Biofilms. Microorganisms. 2023;11(12): 2994.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

Microbial Diversity and Community Structure of Wastewater-Driven Microalgal Biofilms

Dwindling water sources increase the need for efficient wastewater treatment. Solardrivenalgal turf scrubber (ATS) system may remediate wastewater by supporting the developmentand growth of periphytic microbiomes that function and interact in a highly dynamic mannerthrough symbiotic interactions. Using ITS and 16S rRNA gene amplicon sequencing, we profiledthe microbial communities of four microbial biofilms from ATS systems operated with municipalwastewater (mWW), diluted cattle and pig manure (CattleM and PigM), and biogas plant effluentsupernatant (BGE) in comparison to the initial inocula and the respective wastewater substrates.The wastewater-driven biofilms differed significantly in their biodiversity and structure, exhibitingan inocula-independent but substrate-dependent establishment of the microbial communities.The prokaryotic communities were comparable among themselves and with other microbiomes ofaquatic 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 wascharacterized by high biodiversity and many filamentous (benthic) microalgae, the agriculturalwastewater-fed biofilms consisted of less diverse communities with few benthic taxa mainly inhabitedby unicellular chlorophytes and saprophytes/parasites. This study advances our understandingof the microbiome structure and function within the ATS-based wastewater treatment process

Efficiency and biotechnological aspects of biogas production from microalgal substrates

Klassen V, Blifernez-Klassen O, Wobbe L, Schlüter A, Kruse O, Mussgnug JH. Efficiency and biotechnological aspects of biogas production from microalgal substrates. Journal of Biotechnology. 2016;234:7-26.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 has 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

Highly efficient methane generation from untreated microalgae biomass

Abstract 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 abstract Growth condition dependence of anaerobic conversion efficiency of microalgae biomass to methan