Week 3 Lecture October 2001 Metabolism Continued

Lecture Review l Metabolism Basics l Aerobic Metabolism of Organics

This Week’s Lecture l Anaerobic Respiration l nitrate (NO 3 - ), CO 2, sulfate (SO 4 2- ), ferric iron (Fe 3+ ), organics, and others l Fermentation l Syntrophic Association During Conversion of Mixed Acid Products to Methane l Chemolithotrophy l Photosynthesis

Aerobic Respiration Overview l carbon flows to carbon dioxide l electrons flow to external acceptor l energy produced by oxidative phosphorylation through PMF

Respiration of Glucose glucose pyruvate Citric Acid Cycle Electron Transport System CO 2 e-e- ½ O 2 H20H20 glycolysis Electrons flow in the form of reduced dinucleotides (NADH and FADH) ADP ATP GDP GTP

Question? l What happens when the environment is anoxic or anaerobic? l What is the difference between anaerobic and anoxic? l What impact does this have on organic carbon biodegradation? l What is the significance of these changes in environmental management and design?

Anaerobic Respiration l Some bacteria are capable of aerobic respiration and anaerobic respiration (aerobic is preferred due to more favorable energy production) l Other bacteria that carry out anaerobic respiration are obligate anaerobes l In either case, the electron acceptor chosen is based on maximizing free energy production for cell growth

Anaerobic (Anoxic) Respiration of Organics l Organic compounds are most often the original electron donor l Most electron acceptors are inorganics l Electron transport systems in anaerobic respiration is similar to that of aerobic metabolism

Examples of Anoxic Respiration E o ’ NAD + /NADH 2 SO 4 /S 2- ½ O 2 /H 2 0 NO 3 - /NO 2 - Fe 3+ /Fe 2+ Fumarate/Succinate CO 2 /CH 4 S o /HS - increasing energy production l terminal electron acceptors other than oxygen used l less energy produced l carbon flow the same as in aerobic respiration

Nitrate Reduction (Denitrification) l Conversion of nitrate (NO 3 - ) as an electron acceptor to ammonia (NH 4 + ) or nitrate (NO 2 - ) l Nitrite undergoes further reduction to produce nitric oxide (NO), nitrous oxide (N 2 O), and nitrogen gas (N 2 ), all of which are lost to the atmosphere l Denitrification results in a loss of nitrogen from ecosystems and is only carried out biologically by bacteria l Nitrogen removal treatment processes incorporate denitrification

Aerobic Respiration and Denitrification l During aerobic respiration, three areas where H + is pumped out to establish PMF

Denitrification l Only two areas in ETC that pump out H + as compared to three for aerobic respiration l Less energy generated

Methanogenesis and Acetogenesis CO 2 as an electron acceptor CO 2 CH 4 methanogenesis H2H2 acetate acetogenesis

Sulfate Reduction l sulfate (SO 4 )reduction to sulfide (S 2- )requires eight electrons l the first intermediate in this process is the production of sulfite (SO 3 2- ) and requires two electrons l conversion of sulfite to sulfide requires an additional six electrons

Sulfate Dissimilatory Reduction

Why does sulfate inhibit methane formation? l Hydrogen is needed for both processes l Sulfate/sulfide (SO 4 /S 2- ) redox pair has a more positive reduction potential l How would sulfate presence in an anaerobic digester affect methane formation?

Iron Reduction l Ferric iron (Fe 3+ ) reduction to ferrous iron (Fe 2+ ) l Relatively large positive E o ’ indicates that Fe 3+ is an attractive electron acceptor l Ferrous iron is much more soluble and this process has been used in mining iron ore l Because of the high concentrations of iron in some groundwaters, iron reduction is a common reaction in groundwater remediation l Very little Fe 3+ in surface waters at neutral pH

Other Metals as Electron Acceptors l Mn +4 to Mn +2 l important in drinking water and groundwater systems l Cr +6 to Cr +3 l Cr +3 much less toxic and soluble and is precipitated out l AsO 4 3- to AsO 3 3- l mining wastes l SeO 4 2- to SeO 4 2- l major problem in agriculture lands in California

If there are no external electron acceptors?? l Suppose there are no electron acceptors like nitrate, various metals, etc. l What happens to the electrons associated with the organic carbon that is oxidized? l How do cells handle this condition? l What is this called?

Fermentation l organic compounds serve as both e donor and acceptor l no externally supplied e donor l oxidized and reduced products formed l carbohydrates are primary fermentable substrates l ATP production occurs via substrate level phosphorylation

Fermentation l Fermentation reactions are important in: l wastewater treatment processes l phosphorus removal l sludge digestion l BOD removal l wetland systems, especially in bottom l sediments (PCB dechlorination) l agricultural management plans for manure l landfill leachate management

Fermentation Carbon and Energy Flow Organic Compound e donor intermediate intermediate ~P Oxidized Organic intermediate e acceptor Reduced Organic fermentation product electron carrier P ADP ATP substrate level phosphorylation

Pyruvic Acid Fermentation pyruvic acid acetaldehyde + CO 2 ethanol NADH NAD + lactic acid NADH NAD + mixed acids organics NAD + NADH ADP ATP

Mixed Acid Fermentation pyruvic acid formic acid CO 2 H2H2 butyric acid acetic acid propionic acid Complex Organics

Mixed Acid Fermentation l important in the breakdown of organic compounds in anaerobic environments l primary products are organic acids, carbon dioxide, and hydrogen

Conversion of Mixed Acid Fermentation Products to Methane l acetic acid and carbon dioxide are converted to methane in anaerobic environments l hydrogen is consumed in the process l butyric and propionic acid are not converted directly to methane

Methane Formation pyruvic acid formic acid H2H2 butyric acid acetic acid propionic acid CO 2 CH 4

Methane Formation pyruvic acid formic acid CO 2 H2H2 butyric acid acetic acid propionic acid CH 4  G o ’ + CO 2 CH 4

Mixed Acid Conversion to Acetic Acid l Breakdown of acids such as butyric and propionic to acetic is required prior to methane formation l This breakdown is energetically non- favorable at standard conditions l How do organisms alter the environment to achieve this reaction?

Non-Standard Conditions  G =  G o ’ + RT ln ( [C][D]/[A][B] ) l Conversion of butyric and propionic acids results in acetic acid and H 2 l H 2 is consumed by methanogens in the conversion of both acetic acid and CO 2 to methane l The reduction in H 2 makes these reactions possible by lowering the product concentrations in the above equation

Syntrophic Association l Where a H 2 producing organism can only grow in the presence of a H 2 consuming organism l The coupling of H 2 formation and use is called interspecies hydrogen transfer l If H 2 builds up in a process it is indicative of an unbalanced consortium l A H 2 build-up will result in a build up of acids resulting in pH decreases and process failure

Fermentation Summary l Little free energy available for growth l for example in glucose fermentation to ethanol, 2 moles of ATP produced/mole of glucose l Most energy is tied up as products (alcohols, acids, methane, H 2 ) l These products produced as intermediate electron acceptors are reduced l A key intermediate is pyruvate

Can other substances besides organic carbon serve as electron donors?

Chemolithotrophy l the oxidation of inorganics for production of cellular energy l terminal electron acceptor is typically oxygen l most lithotrophs are also autotrophs l accordingly, during lithotrophy there is a need to not only produce energy in the from of ATP but also reduced electron carriers to reduce CO 2 to cell carbon

Electron Flow in Lithotrophs l energy gained from e- flow through ETC is used to drive reverse electron transport against an unfavorable reduction potential to form NADPH and then reduce CO 2 NADPH NADP + Electron Transport Chain e - donor oxid e - donor e-e- O2O2 H20H20 ATP ADP CO 2 CH 2 O e-e- e-e-

Electron Donors for Chemolithotrophy Eo’Eo’ 2H + /H 2 S 0 /HS - SO 4 2- /HS - NH 2 OH/NH ½ O 2 /H 2 0 primary electron acceptor for lithotrophy NO 3 - /NO 2 - Fe 3+ /Fe 2+ l the greater the reduction potential differences between the donor and oxygen, the greater the energy available for growth

Hydrogen Oxidation l chemolithotrophs use hydrogen as an energy source for growth l those bacteria that use hydrogen as an electron donor and oxygen as a terminal acceptor are referred to as hydrogen bacteria (versus methanogens) l Typically these bacteria are autotrophs that convert carbon dioxide to cell carbon via the Calvin cycle. The energy for this comes from oxidation of hydrogen using oxygen as an electron acceptor 6H 2 + 2O 2 + CO 2 CH H 2 0

Sulfur Oxidation l Oxidation of hydrogen sulfide (H 2 S), elemental sulfur (S o ) and thiosulfate (S 2 O 3 2- ) l Final Product is sulfate (SO 4 2- ) l Very important in acid mine drainage, biological corrosion

Sulfur Oxidation Reactions H 2 S + 2O 2 SO H + S o + H /2O 2 SO H + HS - + 1/2O 2 + H + S o + H 2 0 S 2 O H O 2 2SO H + sulfur storage as granules

Iron Oxidizing Bacteria l At neutral pH and ambient conditions, ferrous iron (Fe 2+ ) oxidizes quickly to ferric iron (Fe 3+ ) l Under acid conditions this reaction does not occur spontaneously l Lithotrophs (Iron bacteria )biologically convert Fe 2+ to Fe 3+ under these conditions l Oxidation of iron results in little energy production because reduction potential of to Fe 3+ /Fe 2+ is so close to that of oxygen/water 4Fe 2+ + O H + 4 Fe H 2 0

Nitrification l Conversion of ammonium (NH 4 + ) to nitrate (NO 3 - ) l Nitrite (NO 2 - ) is an intermediate l Nitrification is a very important process agriculturally as it leads to the oxidation of ammonia to nitrate and potential nitrogen loss through denitrificiation l In wastewater treatment, nitrification is often needed to reduce the oxygen demand the effluent

Nitrification Reactions Nitrosomonas NH /2O 2 NO H H + NO /2O 2 NO 3 - Nitrobacter l Oxidation of ammonia results in production of acidic conditions l Very little energy available to nitrifiers because reduction potential relatively close to that of oxygen

Energy Production During Phototrophy l the ability to photosynthesize is based on light sensitive pigments called chlorophylls l all cells have chlorophyll A and typically some others l photosynthesis converts light energy to chemical energy l chemical energy produced is used for cell growth in phototrophs which typically are autotrophs (energy is required to reduce CO 2 to cellular carbon) l photosynthesis occurs in both anaerobic and aerobic environments

Anoxygenic Photosynthesis l energy production in anoxygenic photosynthesis occurs as a result of electron flow through an electron transport chain l electron flow is cyclic l membrane mediated l process is similar to that in aerobic respiration and some electron transport components are common to both systems

Electron Flow and Energy Production in Anoxygenic Photosynthesis Antenna pigment complex P870 Center P870 Center act. Electron Transport Chain e-e- e-e- e-e- ADP ATP PMF Eo’Eo’

Schematic of Anoxygenic Photosynthesis

Photoautotrophs l Phototrophs tend to be autotrophs l As such, there is a need to reduce inorganic carbon (CO 2 ) to organic carbon CO 2 CH 2 O l To reduce CO 2 to organic carbon takes reducing power (NADPH) l Autotrophs use reverse electron transport to produce NADPH reducing power

Use of External Electron Acceptors in Anoxygenic Photosynthesis Antenna pigment complex P870 Center P870 Center act. Electron Transport Chain e-e- e-e- e-e- ADP ATP PMF NADP + e-e- NADPH e-e- H2SH2S

Oxygenic Photosynthesis l involves two distinct but interconnected photoreaction centers l electron flow is non-cyclic l water is the primary electron donor l as water is oxidized, oxygen is liberated l in Eukaryotic organisms oxygenic photosynthesis occurs in chloroplast membranes l in Prokaryotic organism, oxygenic photosynthesis occurs in cytoplasmic membrane

Oxygenic Photosynthesis NonCyclic Electron Flow Photosystem II Photosystem I Electron Transport e-e- e-e- NADP + e-e- NADPH H20H20 1/2O 2 and 2H Eo’Eo’ ADP ATP PMF

Phototrophy Summary l Use of light energy to produce chemical energy in the form of ATP and reduced electron carriers l ATP is used for normal cellular functions and to reverse normal electron flow where necessary to produce reduced electron carriers l reduced electron carriers are used to reduce CO 2 to organic carbon (Calvin Cycle) CO 2 CH 2 O NADPH NADP +