Dr. Warshi Dandeniya

Environmental microbiology

SS 5113 Dr. Warshi Dandeniya Bioenergetics of microorganisms 2 h WS Evolution of metabolic pathways 2 h Reading assignment Techniques in environmental 3 h microbiology Presentation Microbial communities 3 h Biogeocycling of nutrients 4 h Enzymes in the soil env. 4 h Midterm paper

All assessments 60% Microorganisms as sinks and 7 h WB sources of pollutants Presentation (15%) Principles of biological treatments 3 h

End term examination 2h 25% Dr. Warshi Dandeniya Environmental microbiology

The study of microorganisms that inhabit the Earth and their roles in carrying out processes in both natural and human made systems

Ecology Physiology

Environmental Microbial Sciences Ecology Thermodynamics Habitat diversity

Evolution Dr. Warshi Dandeniya Traits of Microorganisms

• Small size • Wide distribution throughout Earth’s habitat • High specific surface area • High rate of metabolic activity • Physiological responsiveness • Genetic malleability • Potential rapid growth rate • Incomparable nutritional diversity • Unbeatable enzymatic diversity

What are the ecological consequences of traits? Can you name major taxonomic groups? Dr. Warshi Dandeniya Ubiquitous in the environment Dr. Warshi Dandeniya So much to explore

• Estimated global diversity of microorganisms ~5 million species

• So far cultured and characterized ~6,500 species

• Documented based on biomarkers ~100,000 species Dr. Warshi Dandeniya An example from polar environment

A metagenomic study with sea water revealed: • Very high microbial diversity in polar ocean • OTUs – 16S RNA gene markers with 97% similarity

Cao et al., 2020 https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-020-00826-9 Dr. Warshi Dandeniya Sri Lankan experience: Diversity of fungi in dry mixed evergreen forests (Dandeniya and Attanayake 2017)

Maximum of 10 different CFU/plate 177 species

Undisturbed forest

Regenerating forest

Chena

8

Culture based approach Metagenomic approach

8 Dr. Warshi Dandeniya

Bioenergetics of microorganisms

Diversity of microbial metabolism Dr. Warshi Dandeniya Metabolism

• The sum of the biochemical reactions required for energy generation and the use of energy to synthesize cell material from small molecules in the environment

Catabolism Anabolism Dr. Warshi Dandeniya Catabolism Anabolism

Energy yielding metabolism Biosynthetic metabolism

Energy sources Biopolymers

Heat ATP

ADP Metabolic products External nutrients Dr. Warshi Dandeniya ATP synthesis

• Substrate level phosphorylation (SLP)

• Electron transport phosphorylation (ETP) Dr. Warshi Dandeniya Substrate level phosphorylation

Simplest, oldest, least evolved Occurs during fermentation, respiration

Substrate (organic) Product (organic) + ATP Dr. Warshi Dandeniya Electron transport phosphorylation

• Complicated, evolved long after SLP • Occurs during respiration, photosynthesis, lithotrophic metabolism, etc. • Requiers: en supply (TCA cycle/photophosphorilation/reduced compound) Membranes Electron transport systems en acceptor ATPase enzymes Dr. Warshi Dandeniya ETP

Electron donor Proton motive force (pmf) e- Electron transport HH++ system (ETS)

ADP

Electron H+ acceptor Membrane bound ATP synthetase ATP enzyme Dr. Warshi Dandeniya Dr. Warshi Dandeniya Diversity of metabolism

• Five groups of organisms classified based on metabolic diversity? Five types of organismsDr. Warshi Dandeniya C Energy Source source Chemical, organic Chemical, Light inorganic Fixed Chemosynthetic Chemosynthetic Photosynthetic organic organoheterotroph lithoheterotroph heterotroph (e.g. humans, (e.g. Thiobacillus (e.g. purple fungi) sp.) and green bacteria)

CO2 Chemosynthetic Photosynthetic lithoautotroph autotroph (e.g. H and S (e.g. plants, oxidizing algae) bacteria Dr. Warshi Dandeniya Diversity of metabolism • Heterotrophic types (Organotrophic) Fermentations Respirations • Lithotrophic types e.g. nitrification, S oxidation • Phototrophic metabolism Oxygenic photosynthesis Anoxygenic photosynthesis Non-photosynthetic photophosphorylation Dr. Warshi Dandeniya Heterotrophic types

Fermentation • Partial oxidation of an organic compound using organic intermediates as en donors and en acceptors • ATP produced by substrate level phosphorylation

• E.g. Homolactic fermentation – Lactobacillus, Streptococci Butyric acid fermentation – Clostridium Butanol-acetone fermentation – C. acetobutylicum Mixed acid fermentation - Enterobacteriaceae Dr. Warshi Dandeniya Dr. Warshi Dandeniya Dr. Warshi Dandeniya Heterotrophic types

Respiration • Complete oxidation of the substrate by an outside en acceptor • ATP produced by SLP & ETP Dr. Warshi Dandeniya Dr. Warshi Dandeniya Lithotrophic type metabolism

• Use of an inorganic compound as energy source • Produce ATP via ETP Dr. Warshi Dandeniya Lithotrophy Physiological group Energy Electron Oxidized end Organisms source acceptor product

Hydrogen bacteria H2 O2 H2O

Methanogens H2O CO2 H2O

- Nitrifiers NH3 O2 NO2 - - NO2 NO3

Methanotrophic CH4 O2 CO2 bacteria 2- Sulfur oxidizers H2S or S O2 SO4

2+ 3+ Iron bacteria Fe O2 Fe Dr. Warshi Dandeniya Phototrophic metabolism

• Use of an electromagnetic energy Convert light energy to chemical energy (ATP) • Produce ATP via ETP and photophosphorylation

• Three types Oxygenic photosynyhesis Anoxygenic photosynyhesis Non-photosynyhetic photophosphorylation Dr. Warshi Dandeniya Significance of the three types of phototrophic type metabolism

Environment with

light , water, oxygen, CO2 (Photosynthetic-autotrophs) light and varying nutrients (Photo-autotrophs, Photo-heterotrophs facultative heterotrophs) light, organic compounds, reduced compounds and ‘No oxygen’ (Photosynthetic- heterotrophs) Dr. Warshi Dandeniya Photosynthesis

Criteria Oxygenic p’sis Anoxygenic p’sis

Organisms Plants, Algae, Purple Sulfur bacteria, Cyanobacteria Green Sulfur bacteria, Purple bacteria, Green bacteria Type of Chlorophyll a bacteriochlorophyll Chlorophyll Absorbs 650 – 750 nm Absorbs 800 – 1000 nm

Produces O2 Yes No

Photosynthetic H2O H2S, other S compounds en donor or organic compounds Non-photosynthetic photophosphorilationDr. Warshi Dandeniya Dr. Warshi Dandeniya Energy yield

• Electron potential of donor and acceptor • Number of electrons transferred

• Further the distance in redox ladder…….. higher the Energy yield Dr. Warshi Dandeniya Energy yield

• Redox reactions: Oxidation is the loss of electrons and reduction is the gain of electrons. Electrons cannot exist in solution and the loss of electrons must be coupled to the gain of electrons

• Reduction potential: This is a quantitative measure of the tendency for a substance to give up electrons in biological systems. It is measured in volts and generally at pH = 7.0 Dr. Warshi Dandeniya Dr. Warshi Dandeniya Relationship between free energy and reduction potential

o DG = -nF D Eh

DGo = Change in Free energy ATP hydrolysis releases –31.8 kJ / mole so we n = number of electrons in need at least this reaction amount of energy to F = Faradays constant make a phosphodiesterase D Eh = E0 (acceptor) - E0 (donar) bond in ATP. Dr. Warshi Dandeniya

Electron Electron Reduced e- Electrode Energy generation donor acceptor end potential (compared to H half cell) product (V) => G0 ’ + H O2 H2O 2 +0.82 -2*96.48*(+0.82- (-0.42)) = - 239 kJ + H CO2 methanol 6 -0.38 - 23.15

H+ Fe3+ Fe2+ 1 +0.76 - 114.33

+ - - H NO3 NO2 2 +0.42 - 162.09 + - H NO3 N2 5 +0.74 - 559.58

+ -2 H SO4 H2S 8 -0.22 - 154.37

H+ Fumarate Succinate 2 +0.03 - 86.83 Range in redox potentials in waterlogged soils and microbiological zoning Dr. Warshi Dandeniya envisioned based on thermodynamic power of en accepting process

+600 2- Oxidized soil O2 à O

+400

- - Moderately NO3 à NO2 , N2O, N2 à 2+ reduced soil +200 MnO2 Mn

2+ Reduced soil 0 Fe2O3 à Fe

2- 2- Highly reduced SO4 à S soil -200 CO2 à CH4 Dr. Warshi Dandeniya Identify the most common electron donor and terminal electron acceptor and calculate the potential energy yield. Predict their habitat.

• Desulfovibrio desulfuricans • Geobacter metallireducens • Thiobacillus denitrificans • Clostridium aceticum • Desulfuromonas acetoxidans • Wolinella succinogens • Nitrosomonas europeae • Pseudomonas aeruginosa • Methanosarcina barkeri