Archaea Biotechnology Dr. Simon K.-M. R. Rittmann  Any technological application using biological systems, living organisms, or derivatives thereof, to manufacture or modify products or processes for specific use. What is Biotechnology? Currently archaea (or componets thereof) are or could be applied in the area of red, white and grey biotechnology Biotechnology Area Application in Green biotechnology Agriculture, plant biotechnology, forestry, food science Red biotechnology Medicine, pharmaceutics, nanobiotechnology White Biotechnology Industrial biotechnology, industrial (bio)chemistry, industrial bioprocessing, biorefinery Grey biotechnology Environmental biotechnology, waste (water) management and treatment, biorefinery, renewable energy production Blue biotechnology Seafood and freshwater food production; supply, safety and control of aquatic organisms Yellow biotechnology Insect biotechnology, food science and technology Archaea Biotechnology 1. Biogas production, anaerobic waste water treatment 2. Bioleaching 3. Nanobiotechnology (S-layer, lipids) 4. Brine treatment (reduction of organic contamination and/or PHA production with extreme halophiles) 5. Utilization of novel (e.g. themotolerant) enzymes 6. Metabolic engineering for CO2 utilization and/or production of specific compounds 7. Biofuel production (e.g. biomethane, biohydrogen) Biofuels Liao et al., 2016renewablegreenenergypower.com Winter, 2000 Global energy systems transition, 1850–2150 The atomic hydrogen to carbon ratio Biofuels: Why? • 1st generation biodiesel • 1st generation bioethanol • 2nd generation • 3rd generation • 4th generation • 5th generation Biofuels Liao et al., 2016 Biodiesel – 1st generation desmoinesregister.com &Eco Energie Etoy alternative-energy-news.info/technology/biofuels/biodiesel-fuel Biodiesel – 1st generation Bioethanol – 1st generation desmoinesregister.com South Plains Ethanol Bioethanol – 1st generation 2nd biofuel generation science.energy.gov.ber 3rd generation biofuels Martinez-Porqueras et al., 2012; Liao et al., 2016 3rd biofuel generation © Pacific Northwest National Laboratory Martinez-Porqueras et al., 2012; Liao et al., 2016, Rittmann, unpublished Bacteria, Archaea (dark fermentation) Volatile fatty acids CO2 H2 4th biofuel generation 5th biofuel generation Martinez-Porqueras et al., 2012; Liao et al., 2016; Rachbauer et al., 2017 Summary biofuels Martinez-Porqueras et al., 2012 Martinez-Porqueras et al., 2012 Summary biofuels Algae, cyanobacteria (biophotolysis) h.ν + 2 H2O O2 2H2 Purple non-sulphur bacteria (photofermentation) h.ν + org. substrate or H2O/CO H2CO2 Bacteria, Archaea (dark fermentation) org. substrate or H2O/CO CO2 H2 hydrogen evolution rate (HER) of 0.1 to 0.4 mol m-³ h-1 HER up to 200 mol m-³ h-1 Biohydrogen production Dark fermentative H2 production Ergal et al. 2018 J Biotech Adv Dark fermentative H2 production Ergal et al. 2018 J Biotech Adv Dark fermentative H2 production Ergal et al. 2018 J Biotech Adv Rittmann et al., 2012 Dark fermentative H2 production Martinez-Porqueras et al., 2013 Dark fermentative H2 production Martinez-Porqueras et al., 2013 Dark fermentative H2 production Martinez-Porqueras et al., 2013 Dark fermentative H2 production Dark fermentative H2 production Ergal et al. 2018 J Biotech Adv Dark fermentative H2 production Ergal et al. 2018 J Biotech Adv • 117 years of dark fermentative H2 production are reviewed (results extracted from 305 papers, the data-set comprised 1732 individual data points) • H2 productivity and Y(H2/S) are compared on C-molar level • The best substrate for H2 production is formate • Thermococcaceae spp. comprise high Y(H2/S) and high qH2 in continuous culture • Thermococcales are the superior organisms for H2 production Strain Isolation site H2 production growth T. gammatolerans Hydrothermal chimney samples from the Guaymas basin + + T. alcaliphilus Shallow marine hydrothermal system from Vulcano − − T. celer Solfataric marine water holes from Vulcano − − T. chitonophagus Hydrothermal vent off the Mexican west coast − − T. profundus Deep-sea thermal vent from the middle Okinawa trench − − T. peptonophilus Izu-Bonin arc − − T. stetteri Marine volcanic crater fields from Kraternya cove − − T. sibiricus Oil wells in western Siberia − − T. onnurineus NA1 Deep-sea hydrothermal vent in the PACMANUS field + + T. barophilus Ch5 Deep-sea hydrothermal field on the Mid-Atlantic Ridge + + Thermococcus sp. DS-1 Hydrothermal field on the East Pacific Rise + + Thermococcus sp. DT-4 Deep-sea hot vents from the southern Pacific basin + + T. litoralis Sh1B Shallow water hot vent off the Kuril Islands − − T. stetteri K1A Shallow water hot vent off the Kuril Islands − − Thermococcus sp. AM4 Deep-sea hot vent on the East Pacific Rise − − Thermococcus sp. Ch1 Hydrothermal structures on the Mid-Atlantic Ridge − − Kim et al., 2010 H2 production by Thermococcus spp. Ergal et al. 2018 J Biotech Adv H2 production from formate H2 production from formate Rittmann et al. 2015 J Biotech Adv H2 production from formate Rittmann et al. 2015 J Biotech Adv Strain Strategy HER [mmol L-1 h-1] Reference Cupriavidus necator ATCC 17699 Immobilization of cells 5.8 Klibanov et al., 1982 Salmonella enterica Closed batch mode 0.3 Pakes and Jollyman, 1901 Escherichia coli SH5 Fed-batch mode with immobilized cells 73.3 Seol et al., 2011 Escherichia coli SR13 hycA disruption and fhlA overexpression 11625 Yoshida et al., 2005 Clostridium butyricum IFO 3847t1 Addition of co-substrate mannitol 0.21 Heyndrickx et al., 1989 Desulfovibrio vulgaris Hildenborough Optimization of reaction conditions 0.67 Martins and Pereira, 2013 Thermococcus onnurineus NA1 Use of high cell density 2820 Lim et al., 2012 HER: hydrogen evolution rate Archaea perform autocatalytic hydrogen production from formate whereas bacteria only perfrom whole cell biocatalysis from formate Rittmann et al. 2012 Microb Cell Fact Rittmann et al. 2015 J Biotech Adv H2 production from formate Reaction equations and their standard Gibbs free energy (G0) for several modes of carboxydotrophic growth Diender et al. , 2015 Isolated microorganisms capable of conserving energy from the water–gas shift reaction H2 production from CO Diender et al. , 2015 Isolated microorganisms capable of conserving energy from the water–gas shift reaction H2 production from CO Rittmann et al. , 2015 H2 production from CO Rittmann et al. , 2015 H2 production from CO Strain Substrate Temp [°C] HER [mmol L-1 h-1] Yield [mol mol-1] Reference Pyrococcus furiosus cellobiose 90 3.8 6.2 Chou et al. 2007 Pyrococcus furiosus maltose 90 2.4 2.4 Chou et al. 2007 Thermococcus onnurineus formate 80 2820 n.a. Lee et al., 2012 Thermococcus onnurineus CO 85 n.a. 1.1 Lee et al., 2012 Thermococcus kodakarensis starch 85 3.9 1.1 Kanai et al., 2005 Thermococcus kodakarensis pyruvate 85 3.2 3.3 Kanai et al., 2005 Desulfurococcus fermentans starch 80 n.a. n.a. Perevalova et al., 2005 Halothermotrix orenii glucose 60 n.a. n.a. Cayol et al., 1994 Methanococcus maripaludis formate 37 n.a. n.a. Lupa et al., 2008 n.a.: not attainable HER: hydrogen evolution rate Rittmann et al. 2012 & 2015 Formate is a cheap feedstock for H2 production, manufactured from e.g. by-product carbon monoxide (CO) of the steel making process Archaeal biohydrogen production Archaeal biohydrogen production Reischl et al. 2018, Folia Microbiol; Reischl et al. 2018 Int J Hydrogen Energ 5th biofuel generation Mauerhofer et al., unpublished H2 CH4 Electrolysis Renewable Energy CO2 Biological CH4 production CO2- containig flue gas Biogas H2-containing flue gas Seifert et al. 2013, Rittmann et al. 2014 4H2 + CO2  CH4 + 2H2O CO2-BMP bioprocessing Rittmann et al., 2015 Scalable BMP process Physiological parameters Bioprocess modes Methanogenic strains Design of bioreactors Media demands CO2-BMP bioprocessing Rittmann et al., 2015 CO2-BMP bioprocessing Rittmann et al., 2015 CO2-BMP bioprocessing Rittmann et al., 2015 CO2-BMP bioprocessing Seifert et al., 2014 CO2-BMP bioprocessing BMP mode MER [mmol L-1h-1] CH4 [Vol.-%] References CSTR 1280 18.3 Nishimura et al., 1992 CSTR 530 96 Peillex et al., 1990 CSTR 950 60 Seifert et al., 2014 Fixed- bed 267 26 Jee et al., 1987 Fixed- bed 228 58 Jee et al., 1988b Hollow fibre 145 14.5 Jee et al., 1988a Trickle bed 1.6* 97.9 Burkhardt and Busch, 2013 * MER calculated per m3 matrix material, MER  methane evolution rate, BMP  biological methane production Rittmann et al. 2015 (a,b) Anaerobic biofilm growing on matrix material for biomethane production in a trickle bed bioreactor. (c) 2L Lab-scale STR-bioreactor for biomethane production.  Either high volumetric productivity (MER) or high methane concentration in the fermentation offgas can be achieved - not both in parallel! (c) Burkhardt et al. 2013 © Simon K.-M. R. Rittmann CO2-BMP bioprocessing Rittmann et al. 2012 CO2-BMP bioprocessing Rittmann et al. 2018 CO2-BMP bioprocessing vvm [L L-1 min-1] D [h-1] qCH4[mmolg-1h-1] Rittmann et al. 2018 CO2-BMP bioprocessing •17800 kWh a-1 (100m2 , 3 persons) Statistik Austria  2.032 kWh h-1 •Biological CH4 production bioreactor produces 950 mmol L-1 h-1 = 212.294 kWh m-3 h-1 A ~10L (C)STR would be sufficient to supply three people living a 100 m2 flat with bioenergy! CO2-BMP bioprocessing  In situ upgrading of biogas-tobiomethane by addition of H2 into the anaerobic digester  Ex situ upgrading of biogas-tobiomethane in a separate bioreactor by contacting H2, biogas and an enrichment culture including hydrogenotrophic methanogens  Ex situ upgrading of biogas-tobiomethane in a separate bioreactor by contacting H2, biogas and a pure culture of hydrogenotrophic methanogens Two principle set-ups for the upgrading of biogas-to-biomethane are indicated. H2 from renewable energy production is converted via water electrolysis (H2 storage tank). The fermentation offgas needs to be analysed regarding the composition of CH4, CO2, H2 (and putatively also H2S). 1a shows in situ biogas upgrading by addition of H2 directly into the anaerobic digester. Due to the simplicity of the set-up a separate bioreactor does not to be included. In 1b the principle set-up for ex situ upgrading of biogas in a separate bioreactor by contacting H2, biogas and an enrichment culture comprising mainly of hydrogenotrophic methanogens or a pure culture of hydrogenotrophic methanogens is used for H2/CO2 conversion. The set-up requires an additional bioreactor but biogas (or also other CO2 or H2 containing industrial flue gasses) can be contacted under defined process conditions as well as by using different type of bioreactors. Rittmann (2015) Advances in Biochemical Engineering/Biotechnology Biogas upgrading Upgrading technology H2 gassing rate [vvm] Stirrer speed [rpm] Temp. [°C] Bioprocess mode, comments Vessel and working volume CH4 offgas [Vol.- %] MER [mmol L-1 h-1] Reference in situ 0.0005 100 55 semi-continuous 4.5 L bioreactor, 3.5 L working volume 65 ± 3.3 0.25 * [18] in situ 0.0012 150 55 semi-continuous, column diffuser 1 L bottle, 0.6 L working volume 53 ± 3 0.56 * [19] in situ 0.0012 300 55 semi-continuous, column diffuser 1 L bottle, 0.6 L working volume 68 ± 2.5 0.66 * [19] in situ 0.0012 150 55 semi-continuous, ceramic diffuser 1 L bottle, 0.6 L working volume 75 ± 3.4 0.69 * [19] ex situ, mixed culture 0.0021 500 55 semi-continuous 1 L bottle, 0.6 L working volume 93.5 ± 4.4 1.35 * [5] ex situ, mixed culture 0.0042 500 55 semi-continuous 1 L bottle, 0.6 L working volume 95.4 ± 2.8 2.74 * [5] ex situ, mixed culture 0.0083 500 55 semi-continuous 1 L bottle, 0.6 L working volume 89.9 ± 4.1 5.25 * [5] ex situ, mixed culture 0.0083 800 55 semi-continuous 1 L bottle, 0.6 L working volume 94.2 ± 2.8 5.39 * [5] ex situ, mixed culture 0.0167 800 55 semi-continuous 1 L bottle, 0.6 L working volume 90.8 ± 2.8 10.59 * [5] ex situ, mixed culture n.a. n.a. 60 continuous culture n.a. n.a. 258.77 * [20] ex situ, mixed culture n.a. n.a. 60 continuous culture, with cell recycle n.a. n.a. 446.15 * [20] ex situ, mixed culture n.a. n.a. 37 continuous culture n.a. n.a. 24.75 * [20] ex situ, mixed culture n.a. n.a. 37 continuous culture, with overpressure n.a. n.a. 40.15 * [20] ex situ, pure culture n.a. n.a. 62 fed-batch n.a. 96 26000 # [21] ex situ, pure culture 0.325 1500 65 chemostat culture, overpressure 10 L bioreactor, 5 L working volume n.a. n.a. [15] ex situ, pure culture 0.067 700 60 chemostat culture bioreactor, 3 L working volume n.a. 23.42 ° [22] ex situ, pure culture 0.533 700 60 chemostat culture bioreactor, 3 L working volume n.a. 50.01 ° [22] ex situ, pure culture 0.067 700 60 chemostat culture bioreactor, 3 L working volume n.a. 22.31 ° [22] n.a.: not attainable * calculated from volumetric H2 uptake rate divided by four # presumably the authors presented total MER (including MER from biogas production) °calculated from volumetric methane production rate Rittmann (2015) Advances in Biochemical Engineering/Biotechnology Biogas upgrading Seifert et al. 2013 (Bio)gas upgrading Seifert et al. 2013 (Bio)gas upgrading Microbial Cell Factories, 2012 International Journal of Hydrogen Energy, 2013 Biotechnology Advances, 2015 International Journal of Hydrogen Energy, 2018 Folia Microbiologica, 2018 Biotechnology Advances, 2018 Further reading – H2 Biomass & Bioenergy, 2012 Critical Reviews in Biotechnology, 2015 AIMS Bioengineering, 2014 Bioresource Technology, 2013 Applied Energy, 2014 Advances in Biochemical Engineering/Biotechnology, 2015 Further reading – CH4 Bioresource Technology, 2017 Frontiers in Microbiology, 2016 Life, 2015 Further reading – CH4 Nature Communications, 2018 Applied Energy, 2018 Applied Microbiology and Biotechnology, 2018 Biotechnology for Biofuels, 2018 Organic Geochemistry, 2018 Chemical Geology, 2018 Folia Microbiologica, 2018