Biogeochemical Cycling in Environmental Microbiology

• Lecture is about Biogeochemical Cycling in Environmental Microbiology
• Instructor: Dr. Nalina Nadarajah, Email: [email protected]

Objective

• Demonstrate and understand carbon and nitrogen cycles, their environmental significance, and the roles of microbes in each cycle

Agenda

• Introduction to Biogeochemical Cycles
• The Carbon Cycle
• The Nitrogen Cycle

Introduction to Biogeochemical Cycles

• Biogeochemical Cycle: A circuit or pathway by which a chemical element or molecule moves through biotic ("bio-") and abiotic ("geo-") compartments of an ecosystem
• All major elements found in biological organisms are cycled
• Understanding these cycles aids the prediction of microbial community development in the environment

Chemical Composition of E. coli Cell Major Elements

• Carbon: 50% of dry mass; a building block of all macromolecules
• Oxygen: 20% of dry mass; a building block of all macromolecules
• Hydrogen: 8% of dry mass
• Nitrogen: 14% of dry mass; found in proteins and nucleic acids
• Sulfur: 1% of dry mass ; Amino acids, vitamins
• Phosphorus: 3% of dry mass; Nucleic acids, ATP

Chemical Composition of E. coli Cell Minor Elements

• Potassium: 2% of dry mass; used for osmotic control
• Calcium: 0.05% of dry mass; provides cell wall stability
• Magnesium: 0.05% of dry mass; an enzyme cofactor
• Sodium: 1% of dry mass; used for osmotic control

Gaia Hypothesis

• Originated by James Lovelock in 1970's and co-developed by Lynn Margulis
• The earth is a super-organism that can respond to drastic environmental changes
• Living organisms and their material environment are tightly coupled and the coupled system is a super-organism. As it evolves there emerges a new property, the ability to self-regulate climate and chemistry

Planetary Atmospheres

• Comparing the atmospheres of Venus, Mars, and Earth, the Earth's atmosphere with life has significantly lower CO2 (0.03%) and substantially higher N2 (78%) and O2 (21%) levels compared to Venus and Mars
• The Gaia hypothesis attributes the drastic atmospheric changes of earth to the development and continued presence of life on earth

Planetary Change

• Microbial and plant activity changed the heat-trapping CO2-rich atmosphere to the present oxygen rich and CO2 poor atmosphere
• This activity lowered Earth's average surface temperature from 290°C to 14.8°C

Development of the Carbon Cycle

• ~3.8 billion years ago, organic carbon was formed by large amounts of UV light reaction with the CO₂ rich atmosphere
• Early heterotrophs used organic matter.
• Microbes developed the ability to fix CO₂ photosynthetically (~3.5 billion years ago)
• Evidence observed from Stromatolites provided a mechanism for carbon recycling
• ~ 2.8 billion years ago photosynthetic microbes developed ability to produce O₂
• Resulting in changes to the atmosphere (accumulation of O₂), the development of the ozone layer, and development of higher forms of life

Carbon Cycle Hypothesis

• The basic carbon cycle involves autotrophs converting CO₂ into organic carbon and heterotrophs converting organic carbon back into CO₂

Potential Issues

• If the earth is a superorganism, it should be able to respond to environmental changes
• The increasing environmental disasters all over the world is concerning.

Global Warming and Greenhouse Gases

• The troposphere (Earth's lower atmosphere, up to 15 km thick) consists of a blanket-like layer of gases keeping Earth warm
• Major traditional gases contributing to heat storage were H₂O (67%) and CO2 (33%)
• Natural and synthetic gases in troposphere have increased during the last century resulting in increased heat trapping
• Natural gases: CO2, methane (CH4), and nitrous oxide (N₂O)
• Synthetic gases: chlorofluorocarbon (CFC), trichlorofluoromethane (CFC-11; freon; CCl3F)

Greenhouse Effect

• The greenhouse effect involves solar radiation passing through the atmosphere, being absorbed by the Earth's surface, and infrared radiation being emitted from the Earth's surface
• Greenhouse gases trap some of this infrared radiation, warming the Earth.

Biogeochemical Cycles

• Biogeochemical cycles were stable for millions of years
• A growing need for food and energy has interfered with these cycles, leading to the formation of greenhouse gases.
• Overall resulting in Global Warming → Climate change

Kyoto Protocol

• The Kyoto Protocol addresses major greenhouse gases: CO2, CH4, N2O, and three groups of fluorinated gases
• Including sulfur hexafluoride (SF6), Hydrofluorocarbons (HFC), and perfluorocarbons (PFC)

Canada and the Kyoto Protocol

• The Kyoto Protocol was established in 1997
• Canada's target was a 6% reduction in GHG by 2012 compared to 1990 levels
• Between 1990 and 2008 - Canada's GHG emission increased by 24%, due to change of government
• The 2011 UN Climate Change Conference in Durban, S.A. – Canada announced its formal withdrawal from Kyoto Accord as of 2012
• Minister of Environment – Peter Kent (2011) stated that, "The Kyoto Protocol does not cover the world's largest two emitters, United States and China, and therefore cannot work"
• Canada has the world's third-largest oil reserves, and the conservative government is reluctant to hurt Canada's booming oil sands sector
• Canada is one of the largest per capital greenhouse gas polluters
• Oil & gas and transportation have large green house gas emissions

Paris Agreement

• Signed in December 2015 in Paris, France
• 195 countries signed including USA, China, Canada & India
• (13 countries (incl. Russia, Iran, Iraq, Turkey remain yet to ratify)
• Former US President Donald Trump announced U.S. would cease all participation in the Paris Agreement
• Current US President Joe Biden has requested UN on rejoining the Paris Agreement

Paris Accord

• Signed in December 2015 in Paris, France
• 195 countries signed including USA, China, Canada & India
• On June 1, 2017, USA announced its withdrawal by 2020
• Canada committed to emissions targets of 17% reduction from 2005 levels by 2020 and 30% by 2030
• Potential solutions include: carbon tax and cap-and-trade

Releasing Greenhouse Gases

• Includes burning of fossil fuels and deforestation - CO2
• Includes manure management, paddy rice farming, wetland changes, and covered vented landfill emissions – CH4
• Includes over use of fertilizers – N₂O

Atmospheric Concentration of Selected GH gasses

• Comparison of pre-industrial and 2004 greenhouse gas concentrations reveals a significant increase in CO2, CH4, N₂O, Sulfur hexaflouride (SF6), and CFC

Greenhouse Effects

• Comparison of greenhouse effect relative to CO₂ reveals that the gases CFC-12, CFC-11 (freon) and Ozone have the highest global warming contributions
• Gases N₂O and CH₄ also have high greenhouse effects

Carbon Cycle facts

• Reservoir: a sink or source of an element
• Global carbon reservoirs include carbonate rock in the earth's crust (1.2 x 1017 metric tons)
• DOM & POM in oceans(2.1 x 1012 metric tons)
• the CO2 in the atmosphere (6.7 x 1011 metric tons)
• The atmospheric reservoir is the most accessible and actively cycled
• The the last 100 years has seen a 28% increase in atmospheric CO₂ likely from human activity
• This increase is responsible, in part, for the Greenhouse Effect and global warming

Carbon Respiration

• CO₂ fixed into organic compounds is consumed by animals & heterotrophic microbes
• End products of respiration are CO2 and new cell mass
• More complex carbon cycle includes anaerobic activity, such as fermentation & methanogenesis
• Aerobic and Anaerobic Respiration transform Carbon Dioxide into Fossil Fuels

Organic Polymers

• Most common organic carbon in the environment are plant polymers, polymers used in bacterial and fungal cell walls, and arthropod exoskeletons
• Includes 3 most abundant polymers - cellulose, hemicellulose, and lignin

Cellulose

• Most abundant polymer found on Earth and makes up the woody structures of plants
• Consists of linear, ẞ-1,4 linked glucose subunits (1000 – 10,000; MW: 1.8 x 106 g/mol)
• Partially degraded by extracellular microbial enzymes (β-1,4-endoglucanase and β-1,4-exoglucanase aka cellulases) before it can be taken up and used by bacteria

Hemicellulose

• The second most common plant polymer with branched and more heterogeneous structure composed of various monosaccharides, including various hexoses, pentoses (~ 200 monomers) & uronic acids
• Contains enzymes E.g. pectin
• It's degradation similar to cellulose; more enzymes are involved

Lignin

• The third most common plant polymer with building blocks in randomly polymerized tyrosine & phenylalanine
• Strengthens cell walls and improves pathogen resistance
• Its Biodegradation is slower and less complete than for other polymers
• Its Extreme heterogeneity makes evolution of specific degradative enzymes difficult broken down somewhat into smaller subunits by H2O2-dependent lignin peroxidase
• Free-radicals generated help break-down
• Subunits taken up by microbes (white rot fungi) and degraded

Methane

• Formed mostly by microbes in an anaerobic process called methanogenesis by methanogens (obligatory anaerobic archae)
• Methanogenesis includes the conversion 4H2 + CO2 -> CH4 + 2H2O
• Occurs in specialized environments like wetlands and paddy fields, landfills and in the rumen gut
• A greenhouse gas that is 25 times more effective than CO2 at trapping heat
• It can be explosive and the generation in landfill sites must be managed (i.e. vented)

Methane Releases and Oxidation

• Methane is released from both biogenic and abiogenic sources.
• Biogenic sources: ruminants, termites, rice paddies, natural wetlands, landfills, oceans, and tundras
• Abiogenic sources: coal mining, natural gas flaring and venting, industrial and pipeline losses, biomass burning, methane hydrates, volcanoes, and automobiles
• Methanotrophs (a.k.a methanophiles) are a group of bacteria oxidize methane as a source of carbon and energy
• Chemoheterotrophic and aerobic, that can couple anaerobic oxidation of methane to nitrite reduction

Methane Monooxygenase

• The first enzyme identified as able to cometabolize highly chlorinated solvents like trichloroethylene (TCE).
• The nonspecific enzyme can oxidize both methane and TCE.
• Subsequent degradation steps are either spontaneous or catalyzed by other bacteria
• Working on strategy to use methanotrophs in bioremediation of contaminated groundwater

Nitrogen Cycle

• The most studied and complex mineral cycle
• Nitrogen is the mineral nutrient in most demand by microbes and plants and is the 4th most common element in cells (12-14% of dry weight)
• Stable valences range from -3 (NH3+) to +5 (NO3¯)
• The Cycle includes microbially-catalyzed processes of nitrogen fixation, ammonification, nitrification and denitrification

Development of the Nitrogen Cycle

• It emerged because nitrogen was a limiting element for microbial growth
• N₂ was abundant in the atmosphere, but inaccessible to cells, as they need organic or reduced inorganic N
• Nitrogen-fixing microbes developed the ability to fix N₂ into organic nitrogen via: the enzyme nitrogenase and the reducing atmosphere found in early Earth

Nitrogen Reservoirs

• Large Reservoirs
  • largely inaccessible and not actively cycled N2(g) in atmosphere (78%)
  • Continually released from volcanic and hydrothermal eruptions and bound ammonium in the Earth's crust
• Small Reservoirs
  • Actively cycled includes organic nitrogen found in living biomass and dead matter
  • Inorganic N ions (ammonium, nitrite and nitrate) are highly water soluble and are distributed throughout the ecosphere

Nitrogen Fixation

• Plants, animals & most microbes require combined forms of nitrogen for growth, but the ability to fix nitrogen is limited to bacteria, archaea, and symbiotic associations
• N fixation – 67% by microbial fixation, 30% by fertilizer production via the Haber-Bosch Process and 3% by atmospheric fixation - lightening
• Fertilizer production is expensive
• Can be substituted with alternatives like rotation of crops between nitrogen-fixers (soybeans) & non-fixers (corn)

Nitrogen Fixation Methods

• Atmospheric fixation by lightning (3%)
  • high energy of lightning breaks nitrogen molecules, combines with oxygen to form nitrogen oxides, dissolves in rain, forms nitrates
• Biological fixation by certain microbes (67%)
  • alone or in a symbiotic relationship with some plants and animals
  • e.g. Cyanobacteria (Anabaena, Nostoc), Azotobacteraceae, Rhizobia, Frankia
• Industrial fixation (Haber-Bosch process) (30%) atmospheric N₂ and H₂ (usually from natural gas or petroleum) can be combined to form NH3 under high P, high T and catalyst

Nitrogen Fixation Chemistry

• Catalyzed by nitrogenase enzyme complex, requires ATP and cytochromes
• N₂ + 8 H+ + 6 e¯ → 2 NH3 + H₂ with AG = +150 kcal/mol
• Involves the incorporation of ammonia into amino acids and nucleic acids
• Regulated via expression of required genes (nif) inhibited by NH3, resulting in Feedback Inhibition
• The enzyme is extremely oxygen sensitive and requires low O₂ tensions to function

Microbes for Nitrogen Fixation

• Free-living soil bacteria like Azotobacter (aerobic), Beijerinckia (aerobic), and Clostridium (anaerobic)
• Azotobacter and Beijerinckia can fix at normal O₂ tension thanks to a mechanisms to protect nitrogenase enzyme
• Rhizobia-legume symbiotic relationships have a rate of fixation is 2-3 orders of magnitude higher than free-living
• Cyanobacteria are predominant in aquatic environments with a fixation rate 1-2 orders of magnitude higher than free-living terrestrial microbes since they are photosynthetic and have specialized heterocysts with thick walls impermeable to O and E.g. Anabaena, Nostoc

Nitrogen Fixation Rates

• Rates vary from Rhizobium-legume which fix 200-300 (kg N/hectare/year)
• Anabaena-Azolla fix 100-120 (kg N/hectare/year)
• Cyanobacteria-moss fix 30-40 (kg N/hectare/year)
• Rhizosphere associations fix 2-25 (kg N/hectare/year)
• Free-living organisms fix 1-2 (kg N/hectare/year)

Nitrogen Fixation Summary

• Is an energy intensive end process with with ammonia as an end-product
• Inhibited by ammonia leading to feed back regulation and occurs in both aerobic and anaerobic environments
• The nitrogenase is O2 sensitive

Ammonium Assimilation (Immobilization)

Incorporation of NH₄⁺ into amino acids (proteins), purines & pyrimidines (nucleic acids) and N-acetylmuramic acid (cell wall)

Ammonification (Mineralization)

Sequential degradation of nitrogenous organic compounds with the release of ammonia

• Proteins → Amino acid → Organic acid + Ammonia
• Under N limiting conditions: immobilization is predominant
• Under N non-limiting conditions: mineralization is predominant
• Fate of ammonium released into the environment can be taken up by plants and microbes or be bound to soil
  • In soil it adds to cation exchange capacity (CEC), trapped in clay, escape to atmosphere, and nitrification

Summary of Ammonia Assimilation and Ammonification

• Assimilation and ammonification cycles ammonia between its organic and inorganic forms
• Assimilation predominates at C:N ratios > 20
• Ammonification predominates at C:N ratios < 20

Nitrification Facts

• Biological oxidation of ammonia to nitrite followed by the oxidation of nitrites to nitrates
• Carried out by a limited number of autotrophic bacteria where the 2 steps are carried out by different populations of bacteria
  • Steps are closely coupled so that build-up of nitrite does not occur
  • pH sensitive and is optimal between 6.6 – 8 and completely inhibited < 4.5
• The oxidation of ammonia to nitrite is carried out by Nitrosomonas and nitrite to nitrate by Nitrobacter or Nitrospira
• Reactions are energy-yielding where nitrifying bacteria use the energy derived from nitrification to assimilate CO₂

Importance of Nitrification to Soil Chemistry

• Transformation of ammonium ions to nitrite and nitrate ions results in a change in charge from '+' to '-'
• Positively charged ions are generally bound by negatively charged clay particles in soil
• Negatively charged ions migrate freely in soil
• Nitrification is therefore ‘nitrogen mobilization’
• Ammonia in soil is rapidly oxidized
  • Nitrate is taken up by plants but can also be leached from the soil into the groundwater, causing eutrophication in lakes
• Health concerns: methemoglobinemia, nitrosamine (carcinogen)

Summary for nitrification

• It is a chemoautotrophic, aerobic process
• It is sensitive to a variety of chemical inhibitors and is inhibited at low pH
• Nitrification in managed systems can result in nitrate leaching and groundwater contamination

Denitrification Facts

• A biologically mediated and more complete reduction of nitrate to nitrogen gas
• Primary denitrifying genera in soil are Pseudomonas and Alcaligenes where NO3 -> NO2 -> NO + N2O -> N2 (g)
• It usually produces a mixture of nitrous oxide and nitrogen often under strictly anaerobic conditions
  • more common in standing waters than in running streams

Denitrification Problems

Removal of limiting nutrient, with N₂O that causes depletion of ozone and is a greenhouse gas

Denitrification Summary

• It is anaerobic respiration using nitrate as TEA and inhibited by oxygen
• Produces a mix of N2 and N2O and many heterotrophs denitrify

Anaerobic Ammonium Oxidation Facts

• A biological process, where ammonium oxidation occurs under anaerobic conditions using nitrite as TEA
  • NH4 + NO2 -> N2 + 2H2O
• Responsible for 50% of the N₂ gas produced in the oceans
• E.g. Brocadia, Kuenenia, Anammoxoglobus which produces hydrazine (rocket fuel; highly toxic) as an intermediate
• Used in the removal of ammonium from wastewater treatment (full-scale plants in the Netherlands)

N Cycle Interruption- Release of N₂O

• Agricultural practices are responsible for large proportion of N₂O released by human activity
  • Such as from ammonia is a primary source of nitrogen in fertilizers and only 50% of applied nitrogen is assimilated by crops
• The rest is lost through leaching, erosion & gaseous emission and may ultimately be released as N₂O
• Other sources of N₂O include burning of biomass as well as combustion of fossil fuel, and chemical manufacturing of nylon

Nitrous Oxide

• Released to the atmosphere from industrial and biological sources
• Contributes to global warming (Greenhouse Effect) & ozone depletion
  • long residence time (114 years) & efficient at radiation absorption (200 X more than CO₂)
  • solar radiation convert N₂O to NO causing factor in O3 depletion

Nitrous Oxide -Photo-dissociation and Ozone Depletion

• With energy N₂O will transform into N₂ + O* as well as NO + O₃ transforming into NO₂ + O₂
• Energy will also transform O₃ into O + O₂ and energy will further transform NO₂ + O transforming into NO + O₂ and finally 2O₃ will turn to + hv -> 3 O₂

Nitrous Oxide & Earth's Atmosphere Cont.

• Is produced by multiple phases of the N cycle
• intermediate in denitrification (wet soil with restricted O₂)
• by-product of nitrification (aerated, moist soils with low O₂)

Nitrate Contamination of Groundwater

• Caused by the Use of fertilizers and large amounts of animal waste leading to excess ammonia in soil & groundwater
• This causes Nitrifying bacteria to convert NH₄ to NO₃⁻; leading to nitrogen accumulation in soil
• Excess nitrate causes methemoglobinemia in infants, and formation of highly carcinogenic nitrosamines in adults

Prevention of Nitrate Contamination

• Best Management Practices (BMPs) for fertilizer amount and timing, as well as irrigation
  • Region-specific since climate and soil types vary by region Use of slow-release fertilizers Application of nitrification inhibitors

Summary

• GH gases are leading to global warming which contribute to climate change
• The UN activities to combat climate change such as with Kyoto Protocol & Paris Agreement
• Carbon cycle -Carbon respiration -Organic carbon polymers -Methane generation & oxidation
• Nitrogen cycle -Nitrogen Fixation -Ammonium Assimilation/ Ammonification -Nitrification
  • Denitrification
  • Anamox