Mp s6
- 1. Session 6 Microbial Cycling of the Elements The sulfur cycle The iron cycle Nutrient-limited growth The chemostat Kinetics of nutrient-limited growth; affinity Mixed-substrate utilisation Mixotrophic growth Microbial Physiology LB2762 1
- 2. The sulfur cycle Microbial Physiology LB2762 reservoir flux 2
- 4. SO4 2- H2S sulfate reduction: anaerobic respiration oxic anoxic Dissimilatory sulfate reduction 4
- 5. Wadlopen: a Dutch pastime Sulfate reduction at work: Iron sulfide in mud from Waddenzee tidal flats 5
- 6. SO4 2- oxic anoxic SH-groups of proteins SH-groups of proteins Assimilatory sulfate reduction many aerobic microorganisms many anaerobic microorganisms 6
- 7. PAPS = phosphoadenosine 5’-phosphosulfate Assimilatory sulfate reduction • Investment of 3 ATP equivalents for sulfate activation • Expensive process (ATP, reducing equivalents) 7
- 8. oxic anoxic SH-groups of proteins SH-groups of proteins Desulfurylation of proteins/amino acids many aerobic microorganisms many anaerobic microorganisms H2S e.g. D-cysteine + H2O → sulfide + NH3 + pyruvate 8
- 9. SO4 2- H2S oxic anoxic hemolithoautotrophic sulfide & sulfur oxidation S0 S0 aerobic oxygen as e-acceptor anaerobic nitrate or Fe3+ as e-acceptor 9
- 10. Sulfur-rich acidic hot spring containing hyperthermophilic H2S and S0 oxidizing Sulfolobus Symbiotic sulfide-oxidizing bacteria living in tube worms 10
- 11. The iron cycle Microbial Physiology LB2762 reservoir flux 11
- 12. 12 The iron reservoirs Natural forms of iron: Fe2+ & Fe3+ (Fe0 mainly anthropogenic) pyrite (FeS2) magnetite (Fe3O4) hematite (Fe2O3) jadeite (Na(Al, Fe)Si2O6) goethite (FeO(OH)) jarosite (HFe3(SO4)2(OH)6)
- 13. 1313
- 14. 14 Bacterial iron reduction chemoorganotrophic iron-reducing bacteria chemolithotrophic iron-reducing bacteria Geobacter metallireducens: Acetate- + 8 Fe3+ + 4 H2O 2 HCO3 - + 8 Fe2+ + 9H+ Geobacter can also use H2 as the electron donor (lithotrophic)
- 15. 15 Bacterial iron oxidation by A. ferrooxidans
- 16. 16 Iron oxidation: bacterial vs chemical
- 17. 17 Iron oxidation: bacterial activity and deposits
- 18. 18 Iron oxidation: biofilms Rio Tinto, Spain
- 19. 19 Iron oxidation: acid mine drainage Acidic mine runoff: pH < 1 Environmental problems (leaching of additional metals)
- 21. 2121
- 22. Nutrient-limited growth Microbial Physiology LB2762 22 All nutrients in excess: batch Nutrient-limited growth: the chemostat Kinetics of nutrient-limited growth; affinity Mixed-substrate utilisation Mixotrophic growth
- 23. Microbial Physiology LB2762 23 All nutrients in excess: batch
- 24. Cx/dt = rx = µ‧Cx (assuming constant volume) = Cx0 ‧eµt Mass balance biomass: change = in - out + production Microbial Physiology LB2762 24 Batch fermentation Doubling time: td = ln(2)/µ (h) Maximum specific growth rate: µmax (h-1 )
- 25. Microbial Physiology LB2762 25 Nutrient depletion/starvation absence/exhaustion of an essential nutrient leads to cessation of growth (µ = 0 h-1 )
- 26. Microbial Physiology LB2762 26 Nutrient depletion in an industrial process: citric acid production by the yeast Yarrowia lipolytica Time (h) glucose ammonia citrate biomass Klasson et al. (1989) Appl. Biochem. Biotechnol. 20:491
- 27. Microbial Physiology LB2762 27 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 2 4 6 8 10 Batch: growth rate profile µ=0 h-1 µ= µmax time µ a rarity in nature: often at least one growth-limiting nutrient
- 28. Microbial Physiology LB2762 28 Kinetics of nutrient-limited growth µ = µ max CS KS + CS original Monod: q = qmax CS KS + CS q1 = specific substrate consumption rate KS = substrate saturation constant CS1 = growth-limiting nutrient concentation q maximum rate Ks 50 % maximum rate S
- 30. Microbial Physiology LB2762 30 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 2 4 6 8 10 µ=0 h-1 µ= µmax time µ
- 31. Microbial Physiology LB2762 31 Nutrient-limited growth: the chemostat chemostat cultivation requires a perfectly mixed culture vessel
- 32. Pump (in and out) Medium reservoir (one single growth limiting substrate) Chemostat Receiving bottle Chemostat – experimental set-up Microbial Physiology LB2762 32
- 33. Microbial Physiology LB2762 33 Chemostat: mathematics Mass balance biomass: change = in - out + production D(VCx)/dt = - ϕv‧Cx + µ‧Cx‧V dCx/dt = (µ - D)‧Cx (at constant volume and with D = ϕv/V) dCx/dt = (µ - D)‧Cx = 0 µ = D (at steady state) With chemostat cultivation the specific growth rate can be set! dilution rate in h-1
- 34. Microbial Physiology LB2762 34 Kinetics of nutrient-limited growth and the chemostat µ = µ max CS KS + CS original Monod: q maximum rate Ks 50 % maximum rate S What is the highest dilution rate at which a steady state can be obtained? Dhighest = µhighest = µ max CS,in KS + CS,in
- 35. Microbial Physiology LB2762 35 Can chemostat cultivation be used as a tool to determine the substrate saturation constant KS? Kinetics of nutrient-limited growth and the chemostat µ = µ max CS KS + CS original Monod: Yes, by accurately measuring the concentration of the limiting nutrient (CS) at varying dilution rates (D=µ). 0 1 2 3 4 5 6 7 8 9 10 0 0.2 0.4 0.6 0.8 1 D CS KS and µ max can be estimated: KS µ max = 0.1 g l-1 = 1.0 h-1 D = 1.0 CS 0.1 + CS This would also work if you plot Cs versus qs.
- 36. Microbial Physiology LB2762 36 The yeast Saccharomyces jansenii is cultivated in a glucose-limited chemostat at a dilution rate of 0.1 h-1 . The glucose concentration in the medium vessel is 10 g/l. From previous studies it is known that the µ max of this organism is 0.4 h-1 and that the KS of this yeast for glucose is 30 mg/l (assuming Monod kinetics of growth). After 5 days a steady state is obtained. a. Calculate the residual glucose concentration in steady state (CS in g/l). b. Calculate the volumetric glucose consumption rate (rS in g/l‧h). Accurately measuring the limiting nutrient? D = µ max CS KS + CS 0.1 = 0.4 CS 30 + CS CS = 10 mg/l = 0.01 g/l rs= glucosein - glucoseout = DCS,in – DCS = 0.1(10-0.01) = 0.999 g/l‧h This means that 0.28 mg glucose per liter per second is consumed. Many organisms even demonstrate a much lower KS for limiting nutrients!
- 37. Microbial Physiology LB2762 37 Measuring the limiting nutrient requires quenching Accurately measuring residual nutrient concentration requires instantaneously stopping the metabolism of that nutrient. Methods for this include: • Liquid nitrogen • Cold inert coolant (e.g. steel balls)
- 38. How do micro-organisms adapt to growth at limiting concentrations of a nutrient? • kinetic adaptation - reduce Ks - increase µmax • adaptation of biomass composition • induction of systems for alternative nutrients 38
- 39. Strategies at low substrate concentration: kinetics decrease Ks µ increase µmax Cs Affinity µmax/KS 39
- 40. Glutamate + NADP NH3 NH3 Glutamate 2-oxoglutarate out in 2-OG + NH3 + NAD(P)H Net Reaction: NADPH NADP N excess GDH Decrease Ks: the ammonia assimilation paradigm Km = 1 – 10 mM 40 2-OG + NH3 + NAD(P)H + ATP NH3 NH3 Glutamine glutamate out in Net Reaction: ATP ADP 2-oxoglutarate NADH NAD 2 glutamate Glutamate + NAD + ADP GOGAT GS N limitation Km = ca. 0.1 mM
- 41. Decrease Ks: potassium uptake by Escherichia coli out in K+ K+ Km ~1 mM K+ excess 41 TrK out in K+ K+ ATP ADP Km ~ 1 µM K+ limitation Kdp
- 42. NADH NAD O2 H2O NUO/ NDH cyt bo Q 2 H+ 2 H+ e- e- O2 excess Decrease Ks: respiration in Escherichia coli NADH NAD O2 H2O NUO/ NDH cyt bd Q 1 H+ 2 H+ e- e- O2 limitation Cytochrome bd oxidase: Low Km for oxygen, but lower H+ pumping efficiency 42
- 43. Adaptive strategies at low substrate concentration decrease Ks µ increase µmax Cs Affinity µmax/KS 43
- 44. Increase capacity: methanol-limited chemostat cultures of Hansenula polymorpha Methanol oxidase: methanol + O2 → formaldehyde + H2O2 First step in methanol metabolism by methylotrophic yeasts methanol oxidase crystalloids 44
- 45. Alternative response to nutrient limitation change biomass composition Reduce content of growth limiting nutrient/element in biomass = Increase biomass yield on growth limiting nutrient/ element 45
- 46. Example: cell wall composition in Bacillus subtilis O O C C C C C COOH OHH H O C C C C C CH2OH NHCOCH3H H O OH H O P excess P limitation Teichuronic acid O O O O O CH2 C C C Ala P OHR O O O O CH2 C C C Ala R O P OH Teichoic acid n Ellwood & Tempest, 1972. Adv. Microbial Physiol. 7:83 46
- 47. Transcription of pyruvate decarboxylase genes in Saccharomyces cerevisiae PDC's 0 500 1000 1500 2000 2500 3000 C-lim N-lim P-lim S-lim levelofexpression PDC1 PDC5 PDC6 mRNA levels (Affymetrix GeneChips) from aerobic, nutrient-limited chemostat cultures (D = 0.10 h-1 ) Viktor Boer et al. (2003) J. Biol. Chem. 278:3265 47
- 48. PDC6 encodes a ‘low-sulfur’ pyruvate decarboxylase PDC6 encodes a ‘low sulfur’ pyruvate decarboxylase that is specifically expressed during S-limited growth Number of sulfur-containing amino acids in the 3 pyruvate decarboxylases of S. cerevisiae Gene Total amino acids Cys Met PDC1 563 4 13 PDC5 563 4 14 PDC6 563 1 5 48 Viktor Boer et al. (2003) J. Biol. Chem. 278:3265
- 49. ‘Sulfur economy’ in Saccharomyces cerevisiae • transcriptome data: shift to ‘low sulfur’ proteins in sulfur- limited cultures • ‘sulfur economy’ is also observed in other microorganisms 49 Viktor Boer et al. (2003) J. Biol. Chem. 278:3265
- 50. Responses of micro-organisms to nutrient limitation induction of genes for alternative pathways Oxygen limitation in facultative anaerobes: induction of pathways for alternative respiration pathways (other electron acceptors) or fermentation: fnr (fumarate-nitrate respiration) in E. coli alcoholic fermentation in bakers’ yeast lactate fermentation in Rhizopus 50
- 51. Responses of micro-organisms to nutrient limitation induction of genes for alternative pathways Induction of systems for uptake and metabolism of less- preferred sources of an element Examples - Induction of amino acid transporters during ammonium- limited growth - Induction of phosphatases during phosphate-limited growth - Induction of sulfatases and systems for cysteine-methionine uptake during sulfate-limited growth 51
- 52. transport SUL2, AGP3, MMP1, MUP1, MUP3, SAM3, GNP1, HGT1, ATM1, COT1, sulfate assimilation SER33, MET1, MET8, MET2, MET3, MET10, MET16, MET22, MHT1, CYS3, STR3 other metabolism ARN1, PDC6, DLD3 regulation MET28, MET32, YOR302W detoxification/stress response GTT2, YHR176W, OYE3, CTT1, RAD59, FLR1, YLL057C other (cell cycle and structure) CSM2, KIC1, ASE1, CWP1 poorly defined/unclassified TIS11, CBP1, SOH1, CHL4, YOL163W, MCH5, YBR293W, YOR378W, YIL166C, YLL055W, BNA3, YJL060W, YFL057C, YLL058W, ICY2, PCL5, YLR364W, YBR281C, YBR292C, YEL072W, YFL067W, YGR154C, YIR042C, YKL071W, YLL056C, YML018C, YNL095C, YNL191W, YOL162W, YOL164W, YPL052W 68 transcripts specifically up-regulated in sulfate-limited cultures of bakers’ yeast (Saccharomyces cerevisiae) low Km sulfate transporter ‘Sulfur Economy’ pyruvate decarboxylase Cys, Met transporterslow Km sulfate transporter Capacity of sulfate assimilation 52
- 53. Strategies for nutrient-limited growth: no free lunch… • Increased Y by changed biomass composition at the expense of decreased functionality (?) • Decreased Ks at the cost of ATP equivalents (energy efficiency) • Increased enzyme synthesis resulting in overcapacity/’metabolic burden’ 53
- 54. Microbial Physiology LB2762 54 So far: Growth on single substrates - one carbon source - one nitrogen source - one sulfur source, etc. How do micro-organisms deal with substrate mixtures?
- 55. Microbial Physiology LB2762 55 Mixed substrate utilization in batch cultures: diauxic growth Mechanism: • Repression by favoured substrate • Induction by less favoured substrate Often at level of transcription, but post-transcriptional mechanisms may also contribute
- 56. Microbial Physiology LB2762 56 Co-consumption is possible at very low concentrations Batch cultivation of E. coli on glucose and galactose at non- Repressing glucose concentration (2 mg/l) glu gal
- 57. Microbial Physiology LB2762 57 Nutrient-limited chemostat cultivation: mixed-substrate utilization at low to intermediate specific growth rates Aerobic chemostat cultivation of Hansenula polymorpha on glucose and methanol at different dilution rates glu biomass methanol • repression at high µ due to ‘Monod’ kinetics
- 58. Microbial Physiology LB2762 58 Mixed-substrate utilization enables growth at lower substrate concentration than on pure substrates Sugar-limited growth of E. coli in chemostat cultures at different glucose-galactose ratios: effect on residual sugar concentrations • important in natural environments • breakdown of pollutants glu gal
- 59. 59 Facultative chemolithoautotrophs: Organic substrates suppress utilization of inorganic electron donor and CO2 fixation Example: Growth of Thiobacillus versutus on acetate and thiosulfate (S2O3 2- ) in aerobic batch cultures
- 60. Biomass yield Sum of autotrophic and heterotrophic biomass yields Rubisco Microbial Physiology LB2762 60 Mixotrophic growth: simultaneous utilization of organic and inorganic carbon sources Chemostat cultivation of T. versutus on thiosulfate-acetate mixtures AUT HET
- 61. Microbial Physiology LB2762 61 Aerobic, respiratory, heterotrophic growth organic substrate organic substrate biomass CO2 CO2 assimilation dissimilation
- 62. Microbial Physiology LB2762 62 Aerobic, respiratory, heterotrophic growth with additional energy source (e.g. thiosulfate) organic substrate organic substrate biomass CO2 CO2 assimilation dissimilation inorganic substrate e.g. sulfate dissimilation
- 63. Microbial Physiology LB2762 63 Aerobic, respiratory, chemolithoheterotrophic growth organic substrate organic substrate biomass CO2 CO2 assimilation dissimilation inorganic substrate e.g. sulfate dissimilation Higher biomass yield than sum of autotrophic and heterotrophic yields!
- 64. Microbial Physiology LB2762 64 Aerobic, respiratory, mixotrophic growth organic substrate biomass CO2 CO2 assimilation dissimilation inorganic substrate e.g. sulfate assimilation biomass
- 65. Biomass yield Sum of autotrophic and heterotrophic biomass yields Rubisco Microbial Physiology LB2762 65 Mixotrophic growth: simultaneous utilization of organic and inorganic carbon sources Chemostat cultivation of T. versutus on thiosulfate-acetate mixtures AUT HET
- 66. Microbial Physiology LB2762 Next lectures: Tuesday June 6 66
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