Jan H. Mussgnug

Microalgae as substrates for fermentative biogas production in a combined biorefinery concept

Most organic matter can be used for bioenergy generation via anaerobic fermentation. Today, crop plants like maize play the dominant role as substrates for renewable biogas production. In this work we investigated the suitability of six dominant microalgae species (freshwater and saltwater algae and cyanobacteria) as alternative substrates for biogas production. We could demonstrate that the biogas potential is strongly dependent on the species and on the pretreatment. Fermentation of the green alga Chlamydomonas reinhardtii was efficient with a production of 587 ml(±8.8 SE) biogas g volatile solids(-1) (VS(-1)), whereas fermentation of Scenedesmus obliquus was inefficient with only 287 ml(±10.1 SE) biogas g VS(-1) being produced. Drying as a pretreatment decreased the amount of biogas production to ca. 80%. The methane content of biogas from microalgae was 7-13% higher compared to biogas from maize silage. To evaluate integrative biorefinery concepts, hydrogen production in C. reinhardtii prior to anaerobic fermentation of the algae biomass was measured and resulted in an increase of biogas generation to 123% (±3.7 SE). We conclude that selected algae species can be good substrates for biogas production and that anaerobic fermentation can seriously be considered as final step in future microalgae-based biorefinery concepts.

An economic and technical evaluation of microalgal biofuels

In her News Feature “Biotech’s green gold”, Emily Waltz details the ‘hype’ being propagated around emerging microalgal biofuel technologies, which often exceeds the physical and thermodynamic constraints that ultimately define their economic viability. Our calculations counter such excessive claims and demonstrate that 22 MJ m−2 d−1 solar radiation supports practical yield maxima of ∼60 to 100 kl oil ha−1 y−1 (∼6,600 to 10,800 gal ac−1 y−1) and an absolute theoretical ceiling of ∼94 to 155 kl oil ha−1 y−1, assuming a maximum photosynthetic conversion efficiency of 10%. To evaluate claims and provide an accurate analysis of the potential of microalgal biofuel systems, we have conducted industrial feasibility studies and sensitivity analyses based on peer-reviewed data and industrial expertise. Given that microalgal biofuel research is still young and its development still in flux, we anticipate that the stringent assessment of the technologys economic potential presented below will assist R&D investment and policy development in the area going forward.

The metagenome of a biogas-producing microbial community of a production-scale biogas plant fermenter analysed by the 454-pyrosequencing technology

Composition and gene content of a biogas-producing microbial community from a production-scale biogas plant fed with renewable primary products was analysed by means of a metagenomic approach applying the ultrafast 454-pyrosequencing technology. Sequencing of isolated total community DNA on a Genome Sequencer FLX System resulted in 616,072 reads with an average read length of 230 bases accounting for 141,664,289 bases sequence information. Assignment of obtained single reads to COG (Clusters of Orthologous Groups of proteins) categories revealed a genetic profile characteristic for an anaerobic microbial consortium conducting fermentative metabolic pathways. Assembly of single reads resulted in the formation of 8752 contigs larger than 500 bases in size. Contigs longer than 10kb mainly encode house-keeping proteins, e.g. DNA polymerase, recombinase, DNA ligase, sigma factor RpoD and genes involved in sugar and amino acid metabolism. A significant portion of contigs was allocated to the genome sequence of the archaeal methanogen Methanoculleus marisnigri JR1. Mapping of single reads to the M. marisnigri JR1 genome revealed that approximately 64% of the reference genome including methanogenesis gene regions are deeply covered. These results suggest that species related to those of the genus Methanoculleus play a dominant role in methanogenesis in the analysed fermentation sample. Moreover, assignment of numerous contig sequences to clostridial genomes including gene regions for cellulolytic functions indicates that clostridia are important for hydrolysis of cellulosic plant biomass in the biogas fermenter under study. Metagenome sequence data from a biogas-producing microbial community residing in a fermenter of a biogas plant provide the basis for a rational approach to improve the biotechnological process of biogas production.

Future prospects of microalgal biofuel production systems.

Climate change mitigation, economic growth and stability, and the ongoing depletion of oil reserves are all major drivers for the development of economically rational, renewable energy technology platforms. Microalgae have re-emerged as a popular feedstock for the production of biofuels and other more valuable products. Even though integrated microalgal production systems have some clear advantages and present a promising alternative to highly controversial first generation biofuel systems, the associated hype has often exceeded the boundaries of reality. With a growing number of recent analyses demonstrating that despite the hype, these systems are conceptually sound and potentially sustainable given the available inputs, we review the research areas that are key to attaining economic reality and the future development of the industry.

Photosynthetic biomass and H2 production by green algae: from bioengineering to bioreactor scale-up

The development of clean borderless fuels is of vital importance to human and environmental health and global prosperity. Currently, fuels make up approximately 67% of the global energy market (total market = 15 TW year(-1)) (Hoffert et al. 1998). In contrast, global electricity demand accounts for only 33% (Hoffert et al. 1998). Yet, despite the importance of fuels, almost all CO(2) free energy production systems under development are designed to drive electricity generation (e.g. clean-coal technology, nuclear, photovoltaic, wind, geothermal, wave and hydroelectric). In contrast, and indeed almost uniquely, biofuels also target the much larger fuel market and so in the future will play an increasingly important role in maintaining energy security (Lal 2005). Currently, the main biofuels that are at varying stages of development include bio-ethanol, liquid carbohydrates [e.g. biodiesel or biomass to liquid (BTL) products], biomethane and bio-H(2). This review is focused on placing bio-H(2) production processes into the context of the current biofuels market and summarizing advances made both at the level of bioengineering and bioreactor design.

Perspectives and advances of biological H2 production in microorganisms

The rapid development of clean fuels for the future is a critically important global challenge for two main reasons. First, new fuels are needed to supplement and ultimately replace depleting oil reserves. Second, fuels capable of zero CO2 emissions are needed to slow the impact of global warming. This review summarizes the development of solar powered bio-H2 production processes based on the conversion of photosynthetic products by fermentative bacteria, as well as using photoheterotrophic and photoautrophic organisms. The use of advanced bioreactor systems and their potential and limitations in terms of process design, efficiency, and cost are also briefly reviewed.

Functional analysis of three type-2 DGAT homologue genes for triacylglycerol production in the green microalga Chlamydomonas reinhardtii.

Photosynthetic organisms like plants and algae can use sunlight to produce lipids as important metabolic compounds. Plant-derived triacylglycerols (TAGs) are valuable for human and animal nutrition because of their high energy content and are becoming increasingly important for the production of renewable biofuels. Acyl-CoA:diacylglycerol acyltransferases (DGATs) have been demonstrated to play an important role in the accumulation of TAG compounds in higher plants. DGAT homologue genes have been identified in the genome of the green alga Chlamydomonas reinhardtii, however their function in vivo is still unknown. In this work, the three most promising type-2 DGAT candidate genes potentially involved in TAG lipid accumulation (CrDGAT2a, b and c) were investigated by constructing overexpression strains. For each of the genes, three strains were identified which showed enhanced mRNA levels of between 1.7 and 29.1 times that of the wild type (wt). Total lipid contents, neutral lipids and fatty acid profiles were determined and showed that an enhanced mRNA expression level of the investigated DGAT genes did not boost the intracellular TAG accumulation or resulted in alterations of the fatty acid profiles compared to wild type during standard growth condition or during nitrogen or sulfur stress conditions. We conclude that biotechnological efforts to enhance cellular TAG amount in microalgae need further insights into the complex network of lipid biosynthesis to identify potential bottlenecks of neutral lipid production.

NAB1 Is an RNA Binding Protein Involved in the Light-Regulated Differential Expression of the Light-Harvesting Antenna of Chlamydomonas reinhardtii

Photosynthetic organisms respond to changes in ambient light by modulating the size and composition of their light-harvesting complexes, which in the case of the green alga Chlamydomonas reinhardtii consists of >15 members of a large extended family of chlorophyll binding subunits. How their expression is coordinated is unclear. Here, we describe the analysis of an insertion mutant, state transitions mutant3 (stm3), which we show has increased levels of LHCBM subunits associated with the light-harvesting antenna of photosystem II. The mutated nuclear gene in stm3 encodes the RNA binding protein NAB1 (for putative nucleic acid binding protein). In vitro and in vivo RNA binding and protein expression studies have confirmed that NAB1 differentially binds to LHCBM mRNA in a subpolysomal high molecular weight RNA–protein complex. Binding of NAB1 stabilizes LHCBM mRNA at the preinitiation level via sequestration and thereby represses translation. The specificity and affinity of binding are determined by an RNA sequence motif similar to that used by the Xenopus laevis translation repressor FRGY2, which is conserved to varying degrees in the LHCBM gene family. We conclude from our results that NAB1 plays an important role in controlling the expression of the light-harvesting antenna of photosystem II at the posttranscriptional level. The similarity of NAB1 and FRGY2 of Xenopus implies the existence of similar RNA-masking systems in animals and plants.

Transcriptome for Photobiological Hydrogen Production Induced by Sulfur Deprivation in the Green Alga Chlamydomonas reinhardtii

ABSTRACT Photobiological hydrogen production using microalgae is being developed into a promising clean fuel stream for the future. In this study, microarray analyses were used to obtain global expression profiles of mRNA abundance in the green alga Chlamydomonas reinhardtii at different time points before the onset and during the course of sulfur-depleted hydrogen production. These studies were followed by real-time quantitative reverse transcription-PCR and protein analyses. The present work provides new insights into photosynthesis, sulfur acquisition strategies, and carbon metabolism-related gene expression during sulfur-induced hydrogen production. A general trend toward repression of transcripts encoding photosynthetic genes was observed. In contrast to all other LHCBM genes, the abundance of the LHCBM9 transcript (encoding a major light-harvesting polypeptide) and its protein was strongly elevated throughout the experiment. This suggests a major remodeling of the photosystem II light-harvesting complex as well as an important function of LHCBM9 under sulfur starvation and photobiological hydrogen production. This paper presents the first global transcriptional analysis of C. reinhardtii before, during, and after photobiological hydrogen production under sulfur deprivation.

THE INTERPLAY OF PROTON, ELECTRON, AND METABOLITE SUPPLY FOR PHOTOSYNTHETIC H2 PRODUCTION IN CHLAMYDOMONAS REINHARDTII

To obtain a detailed picture of sulfur deprivation-induced H2 production in microalgae, metabolome analyses were performed during key time points of the anaerobic H2 production process of Chlamydomonas reinhardtii. Analyses were performed using gas chromatography coupled to mass spectrometry (GC/MS), two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GCxGC-TOFMS), lipid and starch analysis, and enzymatic determination of fermentative products. The studies were designed to provide a detailed metabolite profile of the solar Bio-H2 production process. This work reports on the differential analysis of metabolic profiles of the high H2-producing strain Stm6Glc4 and the wild-type cc406 (WT) before and during the H2 production phase. Using GCxGC-TOFMS analysis the number of detected peaks increased from 128 peaks, previously detected by GC/MS techniques, to ∼1168. More detailed analysis of the anaerobic H2 production phase revealed remarkable differences between wild-type and mutant cells in a number of metabolic pathways. Under these physiological conditions the WT produced up to 2.6 times more fatty acids, 2.2 times more neutral lipids, and up to 4 times more fermentation products compared with Stm6Glc4. Based on these results, specific metabolic pathways involving the synthesis of fatty acids, neutral lipids, and fermentation products during anaerobiosis in C. reinhardtii have been identified as potential targets for metabolic engineering to further enhance substrate supply for the hydrogenase(s) in the chloroplast.