Anaerobic Digestion of the Microalga Spirulina at Extreme Alkaline Conditions: Biogas Production, Metagenome, and Metatranscriptome
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ORIGINAL RESEARCH published: 22 June 2015 doi: 10.3389/fmicb.2015.00597 Anaerobic digestion of the microalga Spirulina at extreme alkaline conditions: biogas production, metagenome, and metatranscriptome Vímac Nolla-Ardèvol 1*, Marc Strous 1, 2, 3 and Halina E. Tegetmeyer 1, 3, 4 1 Institute for Genome Research and Systems Biology, Center for Biotechnology, University of Bielefeld, Bielefeld, Germany, 2 Department of Geoscience, University of Calgary, Calgary, AB, Canada, 3 Microbial Fitness Group, Max Planck Institute for Marine Microbiology, Bremen, Germany, 4 HGF-MPG Group for Deep Sea Ecology and Technology, Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Bremerhaven, Germany A haloalkaline anaerobic microbial community obtained from soda lake sediments was Edited by: Mark Alexander Lever, used to inoculate anaerobic reactors for the production of methane rich biogas. The ETH Zürich, Switzerland microalga Spirulina was successfully digested by the haloalkaline microbial consortium + Reviewed by: at alkaline conditions (pH 10, 2.0 M Na ). Continuous biogas production was observed Aharon Oren, and the obtained biogas was rich in methane, up to 96%. Alkaline medium acted The Hebrew University of Jerusalem, Israel as a CO2 scrubber which resulted in low amounts of CO2 and no traces of H2S Ronald Oremland, in the produced biogas. A hydraulic retention time (HRT) of 15 days and 0.25 g United States Geological Survey, USA Spirulina L−1 day−1 organic loading rate (OLR) were identified as the optimal operational *Correspondence: Vímac Nolla-Ardèvol, parameters. Metagenomic and metatranscriptomic analysis showed that the hydrolysis Institute for Genome Research and of the supplied substrate was mainly carried out by Bacteroidetes of the “ML635J-40 Systems Biology, Center for aquatic group” while the hydrogenotrophic pathway was the main producer of methane Biotechnology, University of Bielefeld, Office G2-152, Universitätstraße 27, in a methanogenic community dominated by Methanocalculus. D-33615 Bielefeld, Germany Keywords: haloalkaline, biogas, methane rich, microalgae, alkaline lake, Spirulina, Methanocalculus [email protected] Specialty section: This article was submitted to Introduction Extreme Microbiology, a section of the journal Extremophilic microorganisms are bacteria and archaea which inhabit, thrive in and colonize Frontiers in Microbiology environments characterized by extremely harsh conditions (Berlemont and Gerday, 2011; Gupta Received: 13 March 2015 et al., 2014). Haloalkaline microorganisms are a specific group of extremophiles which have Accepted: 31 May 2015 the ability to thrive at high concentrations of salt, up to 7.0 M and high pH, up to pH 11, Published: 22 June 2015 and high carbonate concentration (Grant et al., 1990; Baumgarte, 2003; Sorokin et al., 2014). Citation: Their specific abilities to withstand high alkalinity and high salinity have pawned multiple Nolla-Ardèvol V, Strous M and biotechnological applications, such as enzymes for the detergent industry and the bioremediation Tegetmeyer HE (2015) Anaerobic and biotransformation of waste from haloalkaline processes (Horikoshi, 1999; Van Lier et al., 2001; digestion of the microalga Spirulina at Zhao et al., 2014). extreme alkaline conditions: biogas production, metagenome, and Abbreviations: HRT, Hydraulic retention time; OLR, Organic loading rate; OM, Organic matter; CODT, Total chemical metatranscriptome. oxygen demand; CODS, Soluble chemical oxygen demand; VFAs, Volatile fatty acids; SMP,Specific methane potential; BDCH4, Front. Microbiol. 6:597. percentage of substrate conversion to methane; Alk-HRT, Alkaline HRT reactor; Alk-OLR, Alkaline OLR reactor; Alk-Opt, doi: 10.3389/fmicb.2015.00597 Alkaline Optimal reactor; CDS, Coding DNA Sequence; CFB, Cytophaga-Flavobacteria-Bacteroides group. Frontiers in Microbiology | www.frontiersin.org 1 June 2015 | Volume 6 | Article 597 Nolla-Ardèvol et al. Alkaline anaerobic digestion of Spirulina Haloalkaline microorganisms have also been proposed to be between the involved microbial populations can contribute to applied in the production of biofuels such as hydrogen and the optimization of the anaerobic digestion of the desired ethanol (Zhao et al., 2014). Van Leerdam et al. (2008) showed that substrate. Therefore, the metagenome and metatranscriptome it was possible to use a haloalkaline consortium to produce biogas of the haloalkaline anaerobic community responsible for the under controlled conditions. The ability of haloalkaline bacteria degradation of OM and the production of methane is also and archaea to live in alkaline environments could be exploited presented. to produce biogas rich in methane. At alkaline conditions, the − 2− − carbonate system, CO2/HCO3 /CO3 /OH , shifts toward the 2− Materials and Methods formation of bicarbonate, CO3 , therefore, the CO2 released during the decomposition of organic matter (OM) would remain Bioreactor Set-up trapped in solution as (bi)carbonate resulting in biogas composed A 2.0 L semi-continuous stirred tank reactor (S-CSTR) with mainly of methane. This methane rich biogas could directly be a working volume of 1.5 L operating at 35◦C and at high used as biomethane for vehicles or for the national gas grid pH (∼10) and high salt concentration (2.0M Na+) was set (Persson et al., 2006; Weiland, 2010). up and operated at anaerobic conditions. The same reactor In order to produce biogas at alkaline conditions, it is was used in three different experiments: (i) determination of necessary to use a haloalkaline microbial consortium which, the optimal Hydraulic Retention Time (HRT) (Alk-HRT); (ii) for example, can be obtained from soda lakes which are determination of the optimal Organic Loading Rate (OLR) (Alk- natural ecosystems with pH values of up to 12 and high OLR) and (iii) operation at optimal identified parameters (Alk- salt concentrations (Grant, 2006). Some studies have already Opt). The substrate, freeze dried Spirulina (Sonnenmacht GmbH, demonstrated the presence of methanogenic archaea as well −1 Germany) and the alkaline medium, in g L : Na2CO3, 95.0; as the production of methane in soda lakes and in soda lake NaHCO3,15.0;NaCl,16.0andK2HPO4,1.0;werethesameforall sediments (Sorokin et al., 2004, 2015; Nolla-Ardèvol et al., 2012). three experiments. Two different micronutrients solutions were Spirulina is a microalga known to grow in such soda lakes (Jones used throughout the different experiments (Table 1). Solution- and Grant, 1999) and has already been used as substrate for 1 was used in reactors Alk-HRT and Alk-OLR while Solution-2 biogas production at mesophilic pH conditions (Samson and was used in Alk-Opt. The medium was prepared in lots of 1.0 LeDuy, 1986; Varel et al., 1988; Mussgnug et al., 2010). L, its pH was adjusted to 10.0 at 35◦C, and was stored at 37◦C Metagenomics has become a common technique to study until use. Feed was prepared fresh every day by dissolving the taxonomy and gene composition in uncultured microbial appropriate amount of Spirulina in alkaline medium in order communities (Simon and Daniel, 2011). The binning of to obtain the desired organic loading rate. The daily purge and assembled contigs (based on tetranucleotide frequencies) into feed were performed manually with a syringe and through a provisional whole genome sequences can give information about the most abundant and relevant community members (Strous et al., 2012). Moreover, provisional whole genome sequences enable the inference of an ecological function for each major TABLE 1 | Micronutrient solution composition. community member (e.g., biomass hydrolysis, fermentation, Solution-1* Solution-2** methanogenesis etc.). Metatranscriptomics, the sequencing and analysis of mRNAs, can give information about the actual Reactors Alk-HRT/Alk-OLR Alk-Opt active functions of a given microbial community (Gilbert et al., Compound mg L−1 mg L−1 2008; Urich et al., 2008). The combination of the binning FeSO4 · 7H2O – 2000 approach, where provisional whole genome sequences of the FeCl2 · 4H2O 2000 – abundant community members are generated, with the mapping MnCl2 · 4H2O 500 500 of transcriptome reads to these provisional genomes, can give H3BO3 50 300 firsthand information about the ecological function of each of ZnCl2 50 – the most abundant organisms present in a microbial community CoCl2 · 6H2O – 200 (Chistoserdova, 2014). Na2SeO3 · 5H2O 164 164 The anaerobic digestion of OM is a complex process that NiCl2 · 6H2O – 92 involves the participation of both bacteria and archaea (Schlüter ZnSO4 · 7H2O – 100 et al., 2008; Wirth et al., 2012). Under alkaline conditions this AlCl3 · 8H2O – 90 likely also applies but to date, the different functional groups have (NH4)6Mo7O24· 4H2O 50 50 only been addressed individually (Sorokin and Kuenen, 2005b; CuCl2 · 6H2O 38 38 Kivistö and Karp, 2011; Antony et al., 2012; Sorokin et al., 2015). Yeast extract 200 200 In this work we present, to the best of our knowledge, Vitamins RPMI-1640*** 1.0 ml the first study of biogas production from organic biomass + at alkaline conditions (pH ∼10; 2.0M Na ) in a semi- Solution was prepared in 1 L batch and added to the macronutrient solution at a continuous stirred tank reactor inoculated with a strict concentration of 10 mL per liter. * Modified from Vidal et al. (1997). haloalkaline microbial consortium. A good understanding of ** Dr. Dimitry Y Sorokin personal communication. the taxonomic composition and the functional interactions *** Sigma Aldrich. Frontiers in Microbiology | www.frontiersin.org