Article published in BMC Infectious Diseases Journal, an impact factor open access journal

The Sulphur Cycle

THE SULPHUR CYCLE

Sulphur like nitrogen and carbon, is an essential part of all living cells and constitute about 1% of the dry matter of cell. Sulphur containing amino acids are always present in almost all kinds of proteins. Plants can absorb directly the sulphur containing amino acids, e.g., cystine, homocysteine, cysteine and methionine.  Besides S-containing amino acids, it is also an important part of growth factors like thiamin, biotin and lipoic acids. However these amino acids fulfill only a small proportion or requirements for sulphur to the plants.  To fulfill rest of the requirements of plants, sulphur passes through a cycle of transformation mediated by microorganisms. Sulphur compounds involved in the sulphur cycle are H₂S, S°, thiosulphate, sulphite (SO₃^--) and sulphate (SO₄^--). Most common forms of sulphur are H₂S, S° and SO₄^--. The greatest reservoir of sulphur in the biosphere is the sulphate in the oceans. Whereas in the soil,

 it accumulates mainly as a constituent of organic compounds and has to be converted to sulphates to become readily available to the plants. The complete cycling of sulphur is schematically represented and some important steps are discussed as under:

Figure 1: Schematic Representation of Sulphur Cycle (Source Microbial ecology, Heintz and Scott)

1. Source of Sulphur:

The major source of sulphur in marine environment is sulphate. While in the lithosphere, the sulhpur is found as sulphate and iron sulphide (FeS). The metal sulphides (FeS) are readily oxidized to sulphates b y both biological and chemical processes.

FeS      ®        oxidized to      ®        sulphate (SO₄^--) [Biologically and Chemically]

The sulphur can exist in many different oxidation states ranging from -2 to +6:

Form                           Example                     Oxidation state

S^2-                                          Sulphide                                  -2

S°                                Elemental form                       0

S₂O₄                                      Hyposulphite                           +2

SO₃^--                            Sulphite                                   +4

SO₄^--                            Sulphate                                  +6

2. Assimilatory Sulphate Reduction:

In most habitats, sulphates are available to plants and microorganisms which is assimilated into sulphydryl compounds (R-SH) that becomes a part of biomass of living organism. This reduction process in which sulphate becomes biomass is known as Assimilatory Sulphate reduction. Various microorganisms and green photosynthetic plants are involved in the process. Since animals can only uptake the reduced form of sulphur it is an important step to transfer the S into a food chain. The assimilation of inorganic sulphate involves a series of transfer reactions initiated by the reaction of sulphate with ATP to form APS (Adenosine 5 Phosphosulphate) and pyrophosphate (PPi). In second reaction, the APS is converted into PAPS (3-phospho adenosine 5-phosphosulpahte) using one more ATP.

            i. SO₄^-- + ATP             ®                    APS + PPi

                                       ATP sulphurylase

           

ii. APS + ATP             ®                    PAPS + ADP

                                         APS kinase

The active sulphate of PAPS is subsequently reduced to yield sulphite and adenosine 3-5 diphosphate (PAP). Again in the second reduction step sulphides are formed which is immediately incorporated into an amino acid, as a reduced organic S-compound.

iii. 2e^- + PAPS             ®                    SO₃^--  + PAP

                                                NADH-PAPS reductase

           

iv. SO₃^-- + PAP                       ®                    S^2- + 6H⁺ + 6e^-

                                                 NADH- SO₃^—reductase

           

v. S^2- + O-acetylserine             ®                    cysteine + H₂O

3. Release of H₂S

H₂S is release to biosphere by both aerobic and anaerobic processes. They can be either release from decomposition of organic compounds (Desulphurylation) or by reduction of inorganic sulphate (Dissimilatory sulphate reduction).

(A) Degradation of Organic Compounds to Release H₂S (Desulphurylation): Sulphur is released from organic dead matter as H₂S by a process called desulphuration.

(i) Degradation of proteins (proteolysis) liberates amino acids which generally contain sulphur.

Protein =/degradation/=Amino acid

(ii) Enzymatic activity of many heterotrophic bacteria results in the release of H2S from further degradation of sulphur containing amino acids.         

Amino acids (cysteine, methionine, cystine)              ®        H₂S

(B) Dissimilatory sulphate reduction (Sulphate respiration): It is also called sulphate respiration. Sulphates may also be reduced to H₂S by the action of Desulfotomaculum bacteria. The process occurs in anaerobic condition below zero (0) mV redox potential. The process is similar to assimilatory sulphate reduction where intermediates are thiosulphate and tetrathionate. Sulphate is first reduced to H₂S by sulphate reducing microorganisms under anaerobic conditions. Many bacteria including species of Bacillus, Pseudomonas, Desulfovibrio do this work. The mechanism of sulphate reduction to hydrogen sulphide involves, firstly, the reduction of sulphate to sulphite utilizing ATP and, secondly, reduction of sulphite to hydrogen sulphide. The whole mechanism of the reduction of sulphate to hydrogen sulphide by Desulfovibrio desulfuricans, the most important bacterium of this reduction. The only different is the H₂S released in this phenomenon is not incorporated into the biomass as in case of assimilatory sulphate reduction.

SO₄^-- + ATP                ®        APS + PPi       ®        thiosulphate     ®        tetrathionate

Tetrathionate               ®                    H₂S (evaporation)

The major genera which take part in the process are Desulfovibrio, Desulfotomaculum, Desulfomonas, Desulfobacter, Desulfococcus, Desulfonema, Desulfosarcina etc.

Example of sulphate reduction by Desulfotomaculum:

CaSO₄ + 4H₂ =/ Desulfotomaculum /= Ca(OH)₂ + H₂S + 2H₂O

Besides microbiological process, the geochemical processes such as volcanic activities also reduce sulphates into H₂S.

The dissimilatory sulphate reduction process carried out by microbes has an economically important application as it causes corrosion of iron. The reaction mechanisms are shown as;

4Fe + 8H⁺                   ®                    4Fe²⁺ + 4H₂ (Anaerobic polarization)

(H₂ produced protects the iron from further oxidation)

4H₂ + SO₄^--                  ®                    H_2­S + 2H_2­O + 2OH^-

In the presence of H₂S and OH^-, the Fe²⁺ is converted into FeS and Fe(OH)₂

4Fe²⁺ + H_2­S + 2OH^- + 4H_2­O              ®        FeS + 3Fe(OH)₂ + 6H⁺

Final Reaction:

4Fe + SO₄^--  + 2H⁺ + 2H_2­O                 ®        FeS + 3Fe(OH)₂

4. Oxidation of Hydrogen Sulphide (H₂S) to Elemental Sulphur

Hydrogen sulphide undergoes decomposition to produce elemental sulphur by the action of certain photosynthetic sulphur bacteria, e.g., members belonging to the families Chlorobiaceae (Chlorobium) and Chromatiaceae (Chromatium). Example:

CO2 +2H2S ---Photosynthetic sulphur bacteria ---CH2O + H2O +S

Some non-sulphur purple bacteria, e.g., Rhodospirillum, Rhodopseudomonas, and Rhodomicrobium which are facultative phototrophs and grow aerobically in the dark and anaerobically in the light can also degrade H₂S to elemental sulphur

5. Oxidation of Elemental sulphur to Sulphates

Elemental form of sulphur accumulated in soil by earlier described processes cannot be utilized as such by the plants. It is oxidized to sulphates by the action of chemolithotrophic bacteria of the family Thiobacteriaceae (Thiobacillus thiooxidans. Thiobacillus ferroxidans, Thiobacillus denitroficans). Example:

2S + 2H2O + 3O2 ---Thiobacillus thioxidans----2H2SO4
Sulphates are the compounds that can readily be taken by the plants and are beneficial to agriculture in the following three ways:

1.      It is the most suitable source of sulphur and is readily available to plants.

2.      Accumulation of sulphate solubilizes organic salts that contain plant nutrients such as phosphates and metals.

3.      Sulphate is the anion of a strong mineral acid (H₂SO₄) and prevents excessive alkalinity due to ammonia formation by soil microorganisms.

Sulphate is assimilated by plants and is incorporated into sulphur amino acids and then into proteins. Animal fulfill their demand or sulphur by feeding on plants and plant products.

Winogradsky Column: The Winogradsky column, which is named after the Russian Microbiologist Sergei Winogradsky, is a model ecosystem that is used in the study of aquatic and sediment microorganism. A Winogradsky column consists of mud or sediment placed within a glass or clear plastic cylinder. The height of column allows the development of an aerobic zone at the surfaces and microaerophillic and anoxic zones below the surface. The column is exposed to light so that various photosynthetic populations develop at differing depths in the column. Various microbial zones developed in the column as below;

1. Photosynthetic zone: The top layer of the column is the photosynthetic zone. The predominant organisms in this zone are Algae and cyanobacteria. In presence of sunlight, these oraganisms photosynthesize to prepare their own food and oxygen is evolved in the reaction.

CO₂     ®        CH₂O,             H₂O     ®        O₂

                        Carbohydrates

2. Aerobic heterotrophic zone: Beneath the photosynthetic zone there is highly aerated zone. The oxygen and organic compounds in the zone is supplied by photosynthetic zone. A large number of heterotrophic bacteria are present in this zone. Carbohydrates and organic compounds are utilized in this zone.

CH₂O ®        CO₂,                O₂        ®        H₂O

            Carbohydrates

Figure 2: Winogradsky column (source Microbial Ecology; Atlas and Bartha)

3. Microaerophillic zone: Low oxygen level is preferred by sulphide oxidizers such as Beggiatoa and Thiothrix, the gradient organisms with white gray filamentous growth. Non acid tolerant Thiobacilli, Thiobacillus thioparus also grow in this zone.

H₂S      ®        S°                           CO₂     ®        CH₂O,            

                        Elemental sulphur                                     Carbohydrates

4. Facultative anaerobic zone: Organic compounds are utilized in this zone in presence or absence of oxygen. The oxygen level decreases more beneath this zone causing anaerobic zone. Enterobacter, Klebseilla, Citrobacter are predominant in this zone.

CH₂O ®        CO₂ + H₂

Carbohydrates

5. Red Brown zone: The upper portion of sand column is reddish brown with the growth of non-sulfur anaerobic photoheterotrophs (Rhodospirilliaceae). E.g. Rhodospirillum

 CH₂O             ®        H₂                         CO₂     ®        CH₂O

Carbohydrates                                                                        Carbohydrates

6. Red-purple zone: Below the red-brown zone, there is a red-purple zone which indicates the growth of purple sulfur bacteria (Chromatiaceae and Ectothiorhodospiraceae). They are photosynthetic organism. E. g. Chromatium spp. and Ectothiorhodospirillum spp. These organisms oxidize H₂S into elemental sulfur.

H₂S      ®        S°                          CO₂     ®        CH₂O

Carbohydrates                                                                        Carbohydrates

7. Green-gray zone: Even lower. A greenish zone indicates the growth of the green sulfur bacteria (Cholobiaceae). These bacteria grow using sulphide or elemental sulfur as the electron donor. E. g.: Chlorobium spp.

H₂S      ®        S°                          CO₂     ®        CH₂O

Carbohydrates                                                                        Carbohydrates

8. Black zone: The intensely black zone extending upward from the bottom of the column which shows the activity of sulphate reducers. The black coloration is due to metal sulphides principally ferrous sulphide (FeS). Some of sulphate reducers are Desulfovibrio, Desulfotomaculum, Desulfomonas, Desulfobacter, Desulfococcus, Desulfonema, Desulfosarcina etc.

SO₄^-- ®           H₂S                      CH₂O              ®        CO₂

                                                                Carbohydrates

The zone also signifies the fermentative heterotrophic zone:

CH₂O             ®        CO₂ + H₂

Carbohydrates

Nitrogen Cycle

NITROGEN CYCLE-An Important Biogeochemical cycle in Biosphere

Nitrogen forms the main bulk of the atmosphere (78%) as well as the biological systems. Various nitrogenous compounds, e.g. proteins, enzymes, chlorophylls, nucleic acids, etc. play viral roles in the life processes of organisms. The atmospheric nitrogen is chemically inert and is not directly taken by most of the living organisms. The latter, therefore, depend on a source of combined nitrogen or organic nitrogen compounds for their growth. They obtain nitrogenous compounds from soil and organic nitrogen compounds for their growth. They obtain nitrogenous compounds from soil and convert them into essential bimolecular needed for their healthy development. In addition, a part of the great reservoir of the atmospheric nitrogen is converted into an organic form by certain free living microorganism and by plant microorganism association which make it available to the plants. Animals obtain it from plants. The percentage of nitrogen in the atmosphere remains constant by the operation of a nitrogen cycle in nature. Nitrogen is continually entering in the air by the action of denitrifying bacteria and continually returning to the cycle through the action of nitrogen fixing microorganism, lightening, and industrial production of artificial fertilizer. These sequences of changes from free atmospheric nitrogen to fixed inorganic nitrogen, to simple organic compounds, to complex organic compounds in the tissues of microorganisms, plants and animals, and the eventual release of this nitrogen back to atmospheric nitrogen is dealt under the ‘nitrogen cycle’

N in the atmosphere:                                       3800,000 x 10⁹ ton

N in the plant biomass:                                   12 x 10⁹ ton

N in the dead organic matter (on land):          300 x 10⁹ ton

N in the plant biomass (in oceans):                 0.3 x 10⁹ ton

N in the organic matter (in oceans):                550 x 10⁹ ton

1. Dinitrogen Fixation:

Only prokaryotes are capable of dinitrogen fixation.

A. Free living N₂ fixing bacteria:

            Aerobic:                      Azatobacter, Methane oxidizing methylotrophs, Cyanobacteria

            Microaerophillic:         Azospirillum, Rhizobium

            Facultative anaerobes: Enterobacter, Klebseilla

            Anaerobic:                   Clostridium, Phototrophs, Desulfovibrio

B. N₂ fixing symbiotic association:

Symbisis:         Rhizobium, Bradyrhizobium, Frankia

Rhizosphere:   Azospirillum, Azatobacter paspali, Klebseilla

Figure 1: Schematic diagram of Nitrogen cycle

N₂ fixing symbiotic association between root nodules and rhizobia:

It is a very important symbiotic association between leguminous plants with Rhizobium. Two more genera are involved in N₂ fixation are Azorhizobium and Bradyrhizobium. Rhizobium are fast growing organism found in root nodules of Alfalfa, Peas, Clover, other leguminous etc. While Bradyrhizobium are slow growing one found in association with soyabeans, lupine, cowpeas, tropical leguminous plants etc. The Azorhizobium grow with atmospheric nitrogen in its free living state.

Naturally, Rhizobia are free living organisms in soil. They are not directly involved in atmospheric nitrogen fixation. However they can invade root hairs, initiate the formation of a nodule on specific plant and develop nitrogen fixing activity under appropriate condition. The nodule formation is a complex sequence of interactions between rhizobia and plant roots. At the starting of nodule formation, plants secrete flavonoids and isoflavonoids which are attracts particular group of rhizobia by chemotaxis. These chemicals induce the expression of a number of nodulation (nod) genes in rhizobia. The gene codes for the production of enzymes which involved in the biosynthesis of species-specific substituted lipopolysaccharides called nod factors. The factor signals roots and elicit the curling of plant root hairs and division of meristematic cells leading to the formation of nodules.

At the beginning, both Rhizobium and Bradyrhizobium species are attracted by amino acids and dicarboxylic acids presents in the root exudates to their specific legume plants. During nodulation process, tryptophan secreted plant root is metabolized to indole acetic acid (IAA) by the rhizobia. The IAA initiates the curling of root hairs which grow around the rhizobia. Then the bacteria penetrate the soft tissue of plant root developing an infection tube (thread) that is surrounded by cell membrane and cellulosic wall. The infection thread extends to the root cortex region. Within the infection tissue, rhizobia multiply forming unusually shaped and sometimes grossly enlarged cells called bacteroids. The bacteroids produce and contain active nitrogenase enzyme which actually takes part in nitrogen fixation.

Figure 2: Schematic diagram of Nitrogen fixation in Bacteroids

The nitrogenase enzyme system catalyzes the reduction of molecular nitrogen to ammonia. The enzyme system is a complex of dinitrogenase reductase (Fe protein) and dinitrogenase (MoFe protein). Electrons are initially transferred to the dinitrogenase reductase (Fe₄S₄ center), They are then transferred to the P clusters (Fe₄S₄) of the dinitrogenase protein. The P clusters pass the electrons to the iron-molybdenum cofactros (FeMoco; Fe₇S₉Mo-homocitrate) of the dinitrogenase and then on to N₂-H₂ is also evolved in the reaction. Nitrogen fixation brought about by nitrogenase producing bacteria converts atmospheric nitrogen to fixed forms of nitrogen (NH₃, which at physiological pH occurs as NH₄⁺) that can be used by other microorganism, plants and animals.

2. Ammonification:

Ammonia released during aerobic and anaerobic decomposition of organic matter is known as ammonification. However the ammonia released by the process is rapidly recycled by plants and microorganisms. Losses of NH3 by vaporization amount to 15% of the total nitrogen loss, 85% being loss from denitrification. Under anaerobic conditions, NH4+ is a stable compound. Two different steps are involved in ammonification: (a) Proteolysis and (b) Amino acid degradation (Deamination).

(a) Proteolysis: Breakdown of protein into its simpler forms is called proteolysis. A number of bacterial species e.g. Clostridium spp. Pseudomonas, Proteins, Bacillus, and soil actinomycetes, and many fungi are extremely proteolytic. They secrete extracellular enzyme, namely, ‘Proteases’ that convert the protein to smaller unites (peptides) which are then attacked by other proteolytic enzyme, namely ‘peptidases’ resulting ultimately in the release of amino acids. The overall process can be summarized in the form of reactions as follows.

Proteins=/proteses/=Peptides=/peptidase/=Amino acids

(b) Amino acid degradation (Deamination): The release of NH₃ from amino acids occurs by different types of deamination.

1. Oxidative deamination:

Glutamate                   ®                                2-oxoglutamate

                        Glutamate dehydrogenase

This process is most important in amino-acid metabolism.

2. Desaturative deamination:

Aspartic acid                ®                    fumarate + NH₃

                                    aspartase

3. Hydrolytic deamination:

Urea                ®                    NH₃ + CO₂

                        Urease

Urease is repressed by NH₄⁺, but also exists as constitutive enzyme, e.g. Proteus spp. and in Sporosarcina ureae.

At neutral pH, little free NH₃ is present and the ionized from NH₄⁺ prevails. Microoganisms dispose of several reactions for primary NH₄⁺ assimilation.

1. NH₄⁺ + 2-oxoglutarate                    ®                                glutamate

                                                Glutamate dehydrogenase

2. NH₄⁺ + Pyruvate                             ®                                alanine

                                                Alanine dehydrogenase

3. NH₄⁺ + glutamate                           ®                                glutamine

                                                Glutamate synthetase

Glutamine synthetase has a high affinity to NH₄⁺ and operates at extremely low NH₄⁺ concentration (less than 1mM).

3. Nitrification:

Nitrification is the aerobic process in which ammonia is oxidized to nitrite and nitrite to nitrate by the chemolithotrophic nitrifying bacteria. The nitrification process occurs in two stages.

A. Ammonia is first oxidized to nitrite via hydroxylamine. The reaction is catalyzed by oxygenase and hydroxylamine reductase.

NH₃ + O₂ + [O]           ®                    NH₄OH

Ammonia                    oxygenase       hydroxylamine

NH₄OH + O₂                   ®                    NO₂^- + ATPs  

                                    Hydroxylamine   Hydroxylamine reductase     nitrite

Overall reaction:          NH₄⁺ + 3/2O₂              ®        NO₂^- + 2H⁺ +H₂O +276KJ

This first step occurs best at high pH values as enzymes involved prefer non-ionized NH₃ form. The bacteria involve in this step are; Nitrosomonas, Nitrosospira, Nitrosolobus, Nitrosovibrio and Nitrosococcus.

B. In the second step, nitrite is oxidized to nitrate, a stable nitrogen oxide form.

NO₂^- + 1/2O₂               ®        NO₃^- + 73KJ

The bacteria involve in this step are; Nitrobacter, Nitrospina, and Nitrococcus. In addition certain fungi e.g., Cephalosporium, Aspergillus and Penicillium have been reported able to carry out nitrification were discovered to be a biological process by Schloesing and Muntz (1877); Winogradsky isolated the bacteria responsible for biological nitrification in 1890.

The whole process is strict aerobic process and doesn't occur at redox values lower than +200mV (E_o). The optimum pH for nitrification is at neutral pH or slightly alkaline pH condition (pH 7-8). The nitrate is highly soluble and leached out easily. Because of this property, it is a disadvantage for agriculture.

4. Denitrification:

Denitrification refers to the conversion of nitrate (nitrite) into dinitrogen and gaseous oxides of nitrogen (NO, N₂O) by bacterial nitrate respiration. The process occurs under anoxic conditions and is performed by essentially aerobic bacteria. Nitrate serves as an alternative electron acceptor in the absence of molecular oxygen. Denitrification has attracted much interest for several reasons. 1) Loss of fertilizer nitrogen means a decreased efficiency of fertilization. 2) By release in addition to nitrogen of NO and N₂O into the atmosphere, denitrification is involved in reactions that may cause destruction of the ozone layer. 3) Denitrification is the mechanism which balances dinitrogen fixation in the global N cycle. 4) Potential application of the process in the removal of nitrogen from waste materials with high nitrate concentrations.

Nitrate reduction is performed by many bacteria, fungi and by all plants assimilating nitrate as the nitrogen source. In this process, which occurs under both aerobic and anaerobic conditions, NO3- is reduced to NH₃. Contrary to assimilatory nitrate reduction, respiratory (dissmilatory) nitrate reduction is only known in bacteria. NO₃^- is reduced via NO₂^- and NO to N₂O and N₂. By using NO₃^- as an electron sink, the bacteria perform nitrate respiration which serves to generate energy as does O₂ respiration. Representative bacteria producing N₂ or N₂O are found in the genera Bacillus, Pseudomonas, Hyphomicrobium, Spirillum, Moraxella and Thiobacillus (T. denitrificans). The second route of anaerobic nitrate reduction leads to nitrite or ammonia. It occurs in a great number of genera, including Enterobacter, Escherichia, Bacillus, Micrococcus, Mycobacterium, Staphylococcus, Vibrio and Clostridium.

                  +4H                                                       +2H                                   +2H                                      +2H

2HNO₃        ®                      2HNO₂            ®             2NO            ®                  N₂O     ®                N₂

         -2H₂O                                                -2H₂O                                 -H₂O                                     -H₂O

Figure3: Denitrification process

References:
1. Microbial Ecology; Atlas and Barth
2. Microbial Ecology; Heintz and Scoltz