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Sulfur in Agriculture

Article in Revista Brasileira de Ciência do Solo · October 2012

DOI: 10.1590/S0100-06832012000500001

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 SULFUR IN AGRICULTURE 1369

REVISÃO DE LITERATURA

SULFUR IN AGRICULTURE(1)

Adriano Reis Lucheta (2) & Marcio Rodrigues Lambais(3)

SUMMARY

Sulfur (S) deficiency in soils is becoming increasingly common in many areas

of the world as a result of agronomic practices, high biomass exportation and
reduced S emissions to the atmosphere. In this review, the incidence and
commercial exploitation of S pools in nature are discussed, as well as the importance
of S for plants and the organic and inorganic S forms in soil and their
transformations, especially the process of microbiological oxidation of elemental
sulfur (S0) as an alternative to the replenishment of S levels in the soil. The diversity
of S0-oxidizing microorganisms in soils, in particular the genus Thiobacillus, and
the biochemical mechanisms of S0 oxidation in bacteria were also addressed.
Finally, the main methods to measure the S0 oxidation rate in soils and the variables
that influence this process were revised.

Index terms: fertilization, agriculture, microbial diversity, soil fertility.

RESUMO: ENXOFRE NA AGRICULTURA

A deficiência de enxofre (S) nos solos vem se tornando cada vez mais comum em várias
áreas do mundo em razão de práticas agronômicas, alta exportação de biomassa e redução
das emissões atmosféricas. Nesta revisão são abordados a incidência, a exploração comercial
e estoques de S na natureza, a importância do S para as plantas, as formas orgânicas e
inorgânicas no solo e suas transformações, assim como, principalmente, o processo de oxidação
microbiológica do enxofre elementar (S0) como alternativa para a reposição dos níveis de S do
solo. Também é abordada a diversidade de microrganismos oxidantes de S0 nos solos, com
destaque para o gênero Thiobacillus, bem como os mecanismos bioquímicos de oxidação do S0
em bactérias. Por fim, foram revisados os principais métodos para determinação da taxa de
oxidação do S0 nos solos e as variáveis que influenciam esse processo.

Termos de indexação: adubação, agricultura, diversidade microbiana, fertilidade do solo.

(1)
Part of Doctoral Thesis approved by the Graduate Program Agricultural Microbiology of the University of São Paulo - Escola
Superior de Agricultura Luiz de Queiroz. This research was supported by FAPESP. Received for publication in February 10,
2012 and approved im July 24, 2012.
(2)
Post Doctoral Fellow at Molecular Microbiology Laboratory, FAPESP fellow, Soil Science Department, ESALQ/USP. Av. Pádua
Dias, 11. Caixa Postal 9, CEP 13419-900 Piracicaba (SP). E-mail: arlucheta@yahoo.com.br
(3)
Professor, Soil Science Department, ESALQ/USP. CNPq Fellow Researcher. E-mail: mlambais@usp.br

R. Bras. Ci. Solo, 36:1369-1379

1370 Adriano Reis Lucheta & Marcio Rodrigues Lambais

INTRODUCTION activities (Albuquerque et al., 2008; Fonseca & Bacic,

2009).
The importance of sulfur (S) in agriculture has The global supplies of S are estimated to be in the
been recognized for more than a century (Bogdanov, range of 5 billion tons contained in natural gas, oil,
1899; Hart & Peterson, 1911). However, the continual metal sulfides, salt domes, and volcanic deposits,
use of concentrated nitrogen (N) and phosphorus (P) approximately 600 billion tons in coal and pyrobitumen
fertilizer formulations that do not contain S, reduced shale, and virtually unlimited amounts in the form
use of S-containing pesticides, greater export of S from of sulfates such as gypsum or anhydrite (Albuquerque
soil in high crop yields, reduced S input through et al., 2008). The S reserves in Brazil are estimated
rainwater, and the reduction of emissions of S dioxide at 48.5 million tons, or 1.2 % of the world reserves
(SO2) from fossil fuel burning to the atmosphere has (Fonseca & Bacic, 2009). The S main reserves in Brazil
led to an increase of S deficiency in soils (Wainwright, are associated with the refining of petroleum and
1984). In some specific areas, such as the Brazilian natural gas. Additionally, S is found in the form of
cerrado, S deficiency in soils can occur naturally pyrobitumen shale from the Irati formation in the
(McClung & Freitas, 1959; Ribeiro Jr. et al., 2001). Paraná basin, in coal in southern Brazil, especially
A sustainable agricultural management to ensure in Santa Catarina (75 % pyrite and 25 % coal), as a
an adequate S supply for plants requires the by-product from the mining of metal sulfides of zinc,
understanding and quantification of S nickel and gold in Minas Gerais and of copper in Bahia,
transformations in the soil, including the predominant Goiás and Pará, and in its native form in stratiform
microbial processes of S immobilization, sediments (7.1 % S) in Siriri, in the State of Sergipe
mineralization, oxidation, and reduction. The use of (Fonseca & Bacic, 2009).
elemental sulfur (S0) as fertilizer is a cheap alternative According to the Brazilian National Department
to replenish S lost from the soil and allows the of Mineral Production (Fonseca & Bacic, 2009), the
utilization of concentrated commercial forms of N and world production of S in 2008 reached approximately
P. However, S0 replenishment also has to be well- 69 million tons. The countries with the highest
adjusted to meet the plant demand for sulfate (SO42-) production were Canada (13.5 %), the United States
in a short period while avoiding soil and water (13 %), China (12 %), Russia (10 %), Japan (4.5 %),
pollution, since S0 oxidation depends on the microbial Saudi Arabia (4.5 %) and Kazakhstan (4 %). The
diversity and on soil and environmental characteristics Brazilian production of S in the same year was 513
(Wainwright, 1984). thousand tons, accounting for 0.7 % of the world
In this article the occurrence of S stocks in nature, production. Of the S produced in Brazil, 33 % was
the general S transformations in soils and the derived from petroleum refining and bituminous shales
quantification methods are reviewed. In addition, the and 67 % from by-products of mining and metallurgy.
microbiological oxidation of S0 and implications for S0 However, the Brazilian production of S is believed to
as fertilizer are discussed. increase because of the recovery of S forms associated
with petroleum refining and the natural gas basins of
the newly discovered pre-salt reservoirs and because of
public policies requiring the reduction of S content in
SULFUR OCCURRENCE AND fuels. Of all S consumed in the world, 55 % was used
TRANSFORMATIONS for the production of fertilizers. In Brazil, this
percentage exceeds 65 %. Most S is used in the form of
Natural occurrence and commercial sulfuric acid for the solubilization of rock phosphate
exploitation of S and production of ammonium sulfate. In addition, S is
also used in pigments, chemicals, paper and steel
Sulfur, a non-metallic chemical element with the manufacturing, pulp fibers, photography, carbon
molecular formula S8, is naturally present in three disulfide production, insecticides, fungicides, explosives,
forms (alpha, beta and gamma). It is yellow (alpha) rubber vulcanization, and other applications
or pale yellow (beta and gamma), insoluble in water, (Albuquerque et al., 2008). In Brazil, in 2008, 2.1
and with varying solubility in organic solvents million tons of S0 were imported at a cost of 1.03 billion
(Albuquerque et al., 2008). The earth’s crust contains US dollars, aside from 508,000 tons of sulfuric acid,
between 0.06 and 0.10 % of S (Havlin et al., 2005), creating a trade deficit of US$ 1.1 billion (Fonseca &
found in native form in volcanic deposits, bedded Bacic, 2009). The trend of increasing domestic
evaporites and salt domes. Sulfur can also be production and sinking of international prices may
associated with minerals in the form of sulfides such
reduce this trade deficit in the future, however.
as chalcopyrite, pyrrhotite, sphalerite, galena,
arsenopyrite, and pyrite, and sulfates, such as The importance of S for plants
anhydrite, barite and gypsum. Natural gas, oil, coal,
bitumen sands and pyrobitumen shale also contain Sulfur is a vital element for all organisms due to
S. Sulfur can also be found as hydrogen sulfide (H2S) its important role in methionine and cysteine
in anoxic soils and as a by-product of industrial biosynthesis. Cysteine is not only an important

R. Bras. Ci. Solo, 36:1369-1379

 SULFUR IN AGRICULTURE 1371

constituent of proteins, but is also essential to Mineralization and immobilization of S in

determine the structural conformation of proteins and soil
metal binding, and contributes to the catalysis of
According to Kertesz & Mirleau (2004), the S pool
enzymatic reactions (Kertesz et al., 2007). Sulfur is
in the soil is controlled by the balance between
also essential for the synthesis of coenzyme A, which
immobilization of soluble S and mineralization of
is important for fatty acid biosynthesis and oxidation,
organic S. The factors controlling immobilization/
amino acid uptake, oxidation of intermediates of the
mineralization include S concentration in organic
citric acid cycle, and for ferredoxin oxidation, which
matter, moisture, pH, presence of plants, cultivation
is vital in photosynthesis and biological N fixation.
time, type of management and, particularly, the
Furthermore, S is important in vitamin synthesis
microbial diversity and soil enzymatic activity
(Havlin et al., 2005).
(Eriksen et al., 1998; Havlin et al., 2005; Schoenau &
Although S uptake by plant roots occurs Malhi, 2008). In general, in oxic soils, organic matter
preferentially in the form of sulfate (SO42-), S can also with a C:S ratio of >400 induces a net temporary
be absorbed as thiosulfate (S2O32-). Leaves can immobilization of SO42- readily available to plants,
additionally absorb small amounts of SO2 (Havlin et whereas organic residues with a C:S ratio of <200
al., 2005). Vitti et al. (2007) have also reported foliar promote net mineralization. Organic materials with
assimilation of S0 in soybean which, regardless of the a C:S ratio between 200 and 400 result in no net
dose and nature of the source, resulted in increased changes in SO42- concentration in the soil solution
N and S levels in the leaves as compared to S0 supplied (Dick et al., 2008). Sources of mineralizable organic S
to the soil. include animal and vegetable wastes; soil microbial
The S concentration in plant tissues varies between biomass and metabolites, and humus (Schoenau &
0.1 and 0.5 %, with decreasing concentrations in plants Malhi, 2008).
of the orders Cruciferae, Leguminosae and The S mineralization rate is extremely low at
Gramineae, respectively. Symptoms of S deficiency temperatures below 10 °C and above 40 °C, with an
in plants are characterized by reduced plant growth optimum temperature around 30 °C. The optimal
and occurrence of uniform chlorosis on younger leaves moisture content for mineralization is approximately
(Havlin et al., 2005). 60 % of field capacity, with a pH of 6-7. Rhizospheric
microbial activity positively affects mineralization
Organic sulfur in soil when compared to non rhizospheric soil. Although the
Inorganic S is a readily available fraction for root S content in soils can drop dramatically after the first
uptake, but represents on average less than 5 % of cultivation, equilibrium can be reached over time
the total S in the soil. The majority of S (> 95 %) in depending on weather conditions, management
soil is bound to organic molecules and is only indirectly practices and local soil characteristics (Havlin et al.,
available to plants (Kertesz & Mirleau, 2004). 2005). The C:N:S ratio of undisturbed soils is higher
Traditional chemical methods allow for the than that of cultivated soils. The reduction of this
fractionation of soil S in three large fractions of organic relationship after successive cultivation cycles
S: (a) organic S not directly bound to carbon (C), which suggests that S is less mineralizable than C and N
is reduced to H2S by hydriodic acid (HI); (b) organic S (Tabatabai, 1984). However, the use of conservative
directly bound to C (C-S), which is reduced to H2S by management practices, e.g., no-tillage, can avoid
Raney nickel, and (c) residual C bonded S. Organic S nutrient loss, resulting in increased soil organic
not directly bound to C is composed primarily of sulfate matter content and S mineralization rates (Schoenau
esters (C-O-S), such as phenol sulfate, sulfated lipids & Malhi, 2008). The S in the sulfate ester fraction
and sulfated polysaccharides, among others. The can reach up to 60 % of the total soil organic S, and
fraction of organic S directly bound to C consists of can be mineralized by the activity of sulfatases, such
the S-containing amino acids, thiols, disulfides, as arylsulfatase, which is produced by a wide variety
sulfones, and sulfonic acids. The third fraction of of heterotrophic microorganisms, especially
organic S is probably composed of sulfonates, Pseudomonas. Other families of sulfatases have
sulfoxides, and heterocyclic S (Freney, 1967; been identified in soil bacteria, such as
Tabatabai, 1984; Kertesz et al., 2007; Eriksen, 2008). alkylsulfatase, serine-dependent alkylsulfatases and
arylsulfotransferase (Kertesz et al., 2007). However,
More recent studies using sulfur K-edge X-ray
recent studies have shown that although S compounds
absorption near edge spectroscopy (XANES) directly
directly bound to C, e.g., S-containing amino acids,
on soil samples or humic fractions demonstrated the
are more reduced, they are mineralized more rapidly
separation of organic S into the following five fractions
than sulfate ester compounds (Solomon et al., 2003;
based on its oxidation state: two reduced S fractions
Kertesz et al., 2007).
(poly, di, and monosulfides, thiols and thiophenes),
two S fractions in intermediate oxidation states Inorganic sulfur in soil
(sulfoxides and sulfonates) and one fraction containing
highly oxidized S (sulfate esters) (Solomon et al., 2003; In nature, sulfur can be found in different oxidation
Zhao et al., 2006; Kertesz et al., 2007). states ranging from -2 to +6, where sulfide (HS-) is

R. Bras. Ci. Solo, 36:1369-1379

1372 Adriano Reis Lucheta & Marcio Rodrigues Lambais

the most reduced and sulfate (SO42-) is the most extent, volatilization. Volatilization losses may be more
oxidized form (Figure 1). Elemental sulfur (S0) is the relevant in flooded soils due to the microbial reduction
immediate product of hydrogen sulfide (H 2 S) of oxidized S forms to volatile H2S (Eriksen et al.,
oxidation and the most stable form of S (Suzuki, 1998).
1999).
As previously mentioned, the inorganic fraction of
S represents less than 5 % of the total S in soil and is
derived from the mineralization of organic S, MICROBIAL OXIDATION OF S0 IN SOIL
atmospheric inputs (acid rain), pesticides, and mineral
fertilizers. According to Schoenau & Malhi (2008), The genus Thiobacillus
between 1 and 5 % of the total soil organic S can be
The first reported use of S0 in agriculture occurred
converted to SO42- by mineralization. In addition,
in 1877 in South Carolina, in the USA. Charles F.
anthropogenic SO2 emissions into the atmosphere have
Panknin suggested incorporating a mixture
been reduced over the past 30 years in many European
containing 95 parts of bones or finely ground mineral
countries and in the USA (Lehmann, 2008). After the
phosphate with 5 parts of finely powdered elemental
adoption of air pollution control laws in 1990, the
S into the soil to solubilize phosphorus with the sulfuric
amount of sulfuric acid in acid rain in the eastern
acid produced by S0 oxidation. In the same year, he
areas of the USA was reduced by 50 % (Malakoff,
patented his discovery, recorded as part of “Letters
2010).
Patent No. 193,890” (Lipman et al., 1916). According
The main S inputs into the soil are indirect, mainly to Lipman et al. (1916), Panknin had the knowledge
through NPK fertilizers. The most common forms of that S0 in soil was oxidized to SO42- producing sulfuric
sulfate in fertilizers are ammonium sulfate (24 % S), acid. However, he did not realize the process was
single superphosphate (12 % S), gypsum (14-18% S), microbiological.
potassium sulfate (18 % S) and potassium magnesium
sulfate (22 % S). The most concentrated S source is As the field of microbiology expanded, several
S0 (100 % S), either linked to bentonite (90 % S) or important new discoveries related to microbial S
suspended in clay (40-60 % S). Some fertilizers can be metabolism were made. For example, Winogradsky
S-enriched by S0 coating, as in the case of S0 coated (1887) reported that Beggiatoa bacteria were able to
urea (10-20 % S). use H2S as an energy source and to fix atmospheric
CO2. Later, Beijerinck (1904) isolated the S-oxidizing
Losses of inorganic S in soils occur through the bacteria Thiobacillus denitrificans and Thiobacillus
adsorption of sulfate on Fe, Al oxides and clays, thioparus. Armed with this new information, Lipman
leaching, erosion, crop exportation and, to a lesser et al. (1916) tested the oxidation capacity of sterilized
and unsterilized soil samples treated with S0.
Although the responsible organism was not isolated
-2 0 +2 +4 +6 at the time, the authors concluded that the S0
H2S S (SO) SO2 SO3 oxidation process was microbiological.
Waksman & Joffe (1922), Lipman’s colleagues at
Hydrogen Elemental Sulfur Sulfur Sulfur
the New Jersey Agricultural Experiment Station,
sulfite sulfur monoxide dioxide trioxide
isolated the bacteria Thiobacillus thiooxidans from a
HS
-
S8/S0 mixture of soil, rock phosphate and S in inorganic
H2SO2 H2SO3 H2SO4 culture media, attributing an ability to oxidize S0 into
sulfuric acid to this isolate. Starkey (1925) published
Sulfoxylic Sulfurous Sulfuric a detailed study on the physiology and main factors
acid acid acid affecting the oxidative process of the newly discovered
2- 2-
bacterium. In 1935, Starkey isolated Thiobacillus
SO3 SO4 novellus, a new facultative heterotrophic bacterium
Sulfite Sulfate able to use thiosulfate as energy source and in 1951,
another important species of the genus Thiobacillus,
S SO3
2-
T. ferrooxidans, was isolated from an acidic iron-rich
Thiosulfate drainage lake of a coal mine in the USA (Temple &
Colmer, 1951). The new bacterium described was very
S SO32- similar to T. thiooxidans and had an ability to oxidize
Tetrathionate Fe and thiosulfate, but was unable to grow on S0.
S SO3
Although the mixture of rock phosphate and S0
described by Panknin and Lipman was not normally
-2
S Sn Polysulfide
used in agriculture at that time, it stimulated interest
Figure 1. Oxidation state of diverse inorganic sulfur in S transformations and the biochemistry of S-
compounds. Extracted from Suzuki (1999). oxidizing bacteria in soils (Starkey, 1966).

R. Bras. Ci. Solo, 36:1369-1379

 SULFUR IN AGRICULTURE 1373

Whereas T. novellus, T. thioparus and T. Chemolithotrophic and heterotrophic organisms are

denitrificans were normally detected in soils with pH prevalent in well-drained soil (Germida & Janzen,
close to 7.0, T. thiooxidans and T. ferrooxidans were 1993).
rarely detected under the same conditions. According to Czaban & Kobus (2000), bacteria are
Additionally, several studies failed to isolate these more efficient in oxidizing S than fungi in experiments
species from agricultural soils, even after the use of with soils treated with S0 and antibiotics. Wainwright
S0 enrichment procedures (Starkey, 1966). Further & Killham (1980) demonstrated the in vitro ability of
studies regarding S0 oxidation in many countries Fusarium solani to oxidize S0, similar to heterotrophic
showed variable results. For example, whereas T. bacteria in sterile and non-sterile soil samples.
thiooxidans was detected in two thirds of the soil However, F. solani was less efficient in oxidizing S0
samples analyzed in Australia (Vitolins & Swaby, than A. thiooxidans, an obligate chemolithotrophic
1969) and New Zealand (Lee et al., 1987), it was not bacterium. Other fungi species described as being able
detected in soil samples from Canada (Lawrence & to oxidize S0 include Aspergillus niger, Mucor flavus,
Germida, 1991) or Scotland (Chapman, 1990). When Trichoderma harzianum (Grayston et al., 1986),
Vitolins & Swaby (1969) tested the in vitro ability to Saccharomyces, and Debaryomyces (Vitolins &
oxidize S0 and thiosulfate of 206 strains of bacteria Swaby, 1969). By sequencing the internal transcribed
isolated from Australian soils, they predominately spacer (ITS) region, Li et al. (2010) identified 18 fungal
identified facultative chemolithotrophic or isolates of the genera Penicillium, Aspergillus,
heterotrophic organisms rather than Paecilomyces, Fusarium, Bipolaris, and Pleosporales
chemolithotrophic Thiobacillus. with the ability to oxidize S0 in vitro.
With the increasing occurrence of soil S deficiency In the domain Bacteria, the obligate
in large areas of Australia and New Zealand and the chemolithotrophic genera most commonly observed
need for fertilization mainly of pasture lands, Swaby in soil belong to Betaproteobacteria (Thiobacillus) and
(1975) used the same approach as Panknin, but Gammaproteobacteria (Acidithiobacillus).
inoculated the mixture of rock phosphate and S0 with Alphaproteobacteria, such as Paracoccus, are often
T. thiooxidans and pelleted it, naming the product mixotrophic (Ghosh & Dam, 2009). Representatives
Biosuper. Several studies have shown the efficiency of the phyla Actinobacteria (Anandham et al., 2008)
of this preparation, with equal or superior results in and Firmicutes (Jiang et al., 2008) also have the ability
some cases to treatments with soluble phosphate to oxidize reduced forms of S in soils.
fertilizers (Partridge, 1980; Rajan, 1981; Robbins et
al., 1984; Besharati et al., 2007). In Brazil, biofertilizers Paracoccus, which is a Gram-negative, spherical,
containing rock phosphate, S0 and T. thiooxidans have facultative chemolithotrophic, aerobic bacterium, but
been applied to various crops, including cowpea, capable of reducing nitrate under heterotrophic
sugarcane, grapes, and melon (Stamford et al., 2003; anaerobic growth and of oxidizing reduced S
Stamford et al., 2007; Moura et al., 2007; Stamford et compounds, is one of the most well-studied genera of
al., 2008) showing similar results as with soluble S S oxidizers (Friedrich et al., 2001; Friedrich et al.,
sources. 2005; Ghosh et al., 2006). Although mostly isolated
from anaerobic digesters and activated sludge,
In 2000, a reclassification of the 17 species of representatives of this genus have been isolated from
the genus Thiobacillus was proposed, based on the rhizospheric soils (Ghosh at al., 2006). One soil isolate
sequence of the 16S rRNA gene and DNA-DNA from India able to oxidize thiosulfate in both mineral
hybridization (Kelly & Wood, 2000). The new and organic culture media showed high similarity to
classification proposed three new genera Paracoccus versattus and P. alcaliphilus, based on
(Acidithiobacillus, Halothiobacillus and the sequence of the 16S rRNA gene (Deb et al., 2004).
Thermithiobacillus) and the reclassification of other Bacterial isolates from the rhizospheric soil of
species in the existing genera. T. thiooxidans and leguminous plants similar to P. bengalensis, P.
T. ferrooxidans (Gammaproteobacteria) were pantotrophu and P. thiocyanatus were able to oxidize
renamed Acidithiobacillus thiooxidans and different forms of S, including thiosulfate,
Acidithiobacillus ferrooxidans, whereas T. novellus tetrathionate, thiocyanate, sulfide and elemental S
(Alphaproteobacteria) was renamed Starkey novella (Ghosh et al., 2006; Ghosh & Roy, 2007b).
(Robertson & Kuenen, 2006).
Recent studies have reported the isolation of novel
Diversity of S-oxidizing soil microorganisms mixotrophic genera of bacteria capable of oxidizing
reduced forms of S in the soil, including genera before
Reduced S forms can be oxidized in the soil by associated with S oxidation. El-Tarabily et al. (2006)
chemolithotrophic microorganisms that derive energy published the first report on the isolation of Rhizobium
from the oxidation of inorganic compounds and use spp. strains able to oxidize S0 in calcareous soils from
CO2 as carbon source, such as A. thiooxidans, the United Arab Emirates. Another nitrogen-fixing
anoxygenic photoautotrophic (purple and green sulfur bacterium isolated from the rhizosphere of a
bacteria) and heterotrophic bacteria and fungi that herbaceous legume, Mesorhizobium thiogangeticum,
derive carbon and energy from organic substances. has also been described as capable of oxidizing S0 and

R. Bras. Ci. Solo, 36:1369-1379

1374 Adriano Reis Lucheta & Marcio Rodrigues Lambais

thiosulfate (Ghosh & Roy, 2006). New Azospirillum protein, and sox(CD) 2 genes encode a sulfur
and Pseudoxanthomonas strains were also isolated dehydrogenase (Ghosh & Dam, 2009). The Sox
from legume rhizosphere (Ghosh & Roy, 2007a). complex covalently binds to a molecule of thiosulfate
Stubner et al. (1998) isolated strains of facultative by means of a cysteinyl residue located at the C
chemolithotrophic Ancylobacter aquaticus, terminus of the SoxY subunit of the SoxYZ protein
Xanthobacter sp, Bosea thiooxidans and the obligate and oxidizes the S atoms by transferring electrons to
chemolithotrophic Thiobacillus thioparus from the a c-type cytochrome without the formation of
rhizosphere of rice. Other species isolated and intermediate compounds. Other forms of S are
associated with S oxidation in rice fields were introduced into the system in the relative position of
phylogenetically related to Mesorhizobium loti, its oxidation state through enzymatic or non-
Hydrogenophaga sp., Delftia sp, Pandoraea sp., enzymatic conjugation with SoxY (Ghosh & Dam,
Achromobacter sp (Graff & Stubner, 2003) and 2009). The sox operon is widely distributed within
Methylobacterium oryzae (Anandham et al., 2007). the domain Bacteria; the occurrence and evolution of
Heterotrophic, chemolithotrophic and mixotrophic this enzyme complex in nature has been evaluated
growth of Burkholderia and Alcaligenes predominantly through amplification and phylogenetic
(Alphaproteobacteria), Pandoraea (Betaproteobacteria), analysis of the soxB gene (Ghosh et al., 2001; Meyer
Dyella and Halothiobacillus (Gammaproteobacteria) and et al., 2007; Anandham et al., 2008).
Microbacterium and Leifsonia (Actinobacteria), on
thiosulfate, was also described (Anandham et al., 2008). The S4I route, with the formation of tetrathionate
as an intermediate in the oxidation of thiosulfate is
Mechanisms of S oxidation in bacteria not yet as well understood as the Sox pathway due to
the controversial identification of several proteins of
Thiosulfate is an important reduced form of S the system (Ghosh & Dam, 2009). The most recently
present in different environments; it is used as a proposed mechanism was described for the bacterium
source of electrons for energy generation in Tetrathiobacter kashmirensis (Dam et al., 2007). In
photosynthetic and respiratory systems of a wide this case, a periplasmic oxidation system performs
variety of bacteria. Another reduced form of S widely the oxidation of thiosulfate to tetrathionate, whereas
used by chemolithotrophic bacteria is tetrathionate, the complete oxidation of tetrathionate occurs on the
which can also be produced as an intermediate membrane. Sulfite is oxidized in the cytoplasm by a
sulphur compound in the oxidation of thiosulfate sulfite dehydrogenase, which involves a ubiquinone-
(Ghosh & Dam, 2009). Over time, two oxidation
cytochrome b-mediated transfer of electrons to oxygen
processes for sulfide, thiosulfate and elemental S have
(Dam et al., 2007; Ghosh & Dam, 2009).
been proposed, one involving the formation of
tetrathionate as intermediate sulphur compound and
the other involving the direct oxidation of thiosulfate
to sulfate (Kelly et al., 1997).
METHODS FOR DETERMINATION OF S0
Several obligate chemolithotrophic Beta- and OXIDATION IN SOIL
Gammaproteobacteria, such as Acidithiobacillus,
produce tetrathionate as intermediate (S4I), whereas
photo and chemolithotrophic Alphaproteobacteria, According to Wainwright (1984), the majority of
such as Paracoccus, use the mechanism known as the studies focusing on S0 oxidation involve the
“Paracoccus sulfur oxidation” or “PSO pathway” also incubation of soil samples with S0 under laboratory
known as Kelly-Friederick pathway, which is conditions for several weeks, followed by the analysis
controlled by the sox operon (Kelly et al., 1997; of ionic S species and/or changes in the population of
Friederich et al., 2001; Friederich et al., 2005). A third S-oxidizing bacteria. The S0 oxidation rate in the soil
mechanism of thiosulfate oxidation in anaerobic photo can be evaluated and determined directly by
or chemolithotrophic bacteria that deposits sulfur quantifying the S0 remaining after the incubation
intracellularly is known as the “branched thiosulfate period (Watkinson et al., 1987; Watkinson & Lee,
oxidation pathway” (Ghosh & Dam, 2009). 1994) or indirectly based on the quantification of
sulfate formed as a final product of S0 oxidation
The Sox pathway is the most studied and well (Massoumi & Cornfield, 1963; Janzen & Bettany,
understood S oxidation pathway, even though several 1987). Other indirect measurement methods include
steps are still unclear, mostly in relation to the pH determination, which tends to decrease with the
oxidation of S0. In this process, a multienzyme complex,
formation of sulfuric acid (Massoumi & Cornfield,
encoded by the soxTRS-VW-XYZABCDEFGH operon
1963), respirometry (Baldensperger, 1976), and 14CO2
drives the oxidation of sulfide, thiosulfate, sulfite and
uptake (Belly & Brock, 1974).
S0 (Mukhopadhyaya et al., 2000; Bagchi & Roy, 2005;
Lahiri et al., 2006; Ghosh et al., 2009). The soxXA The remaining S0 can be determined by high
genes encode a c-type cytochrome, soxYZ genes encode pressure liquid chromatography (HPLC) analysis of
proteins that covalently bind sulfur and sulfur chloroform soil extracts, using a reverse phase column
compound-chelating proteins, respectively, soxB and an isocratic gradient of methanol and chloroform
encodes a monomeric, dimanganese-containing (Lauren & Watkinson, 1985; Watkinson et al., 1987).

R. Bras. Ci. Solo, 36:1369-1379

 SULFUR IN AGRICULTURE 1375

Several variations of the HPLC method for 1967). Nor & Tabatabai (1977) observed an increase
determination of S0 in hydrocarbons, soil, metal in the oxidation rate with an increase in incubation
sulfides and water have been published (Clarck & temperature of soils in Iowa. The average percent of
Lesage, 1989; Rethmeier et al., 1997; McGuire & S0 oxidation after 57 days of incubation was 8 % at 5
Hamers, 2000; Hurse & Abeydeera, 2002). A gas °C, 22 % at 15 °C and 47 % at 30 °C.
chromatography method has also been described as an
The relationship between S0 oxidation and soil
alternative for S0 quantification in soil samples
moisture is parabolic, with minimal oxidation
(Richard et al., 1977). The amount of S0 dissolved in
occurring when water availability is low, rising when
acetone can also be determined using turbidimetry after
replacing the solvent with water, dispersing the colloidal moisture availability increases up to a peak and then
S0 and determining absorbance at 420 nm (Hart, 1961). decreasing again when the moisture content exceeds
The S0 can also be extracted from soil with chloroform, optimal levels (Janzen & Bettany, 1987). The optimum
treated with HNO 3 -KNO 3, and determined by moisture for maximum S0 oxidation is near the soil
inductively coupled plasma atomic emission field capacity since it allows for good soil aeration
spectroscopy (ICP-AES) (Zhao et al., 1996) or sulfur K- (Wainwright, 1984). Solberg et al. (2005) recovered
edge X-ray absorption spectroscopy (Burton, 2009). between 32 and 53 % and between 72 and 106 % of
the soil SO42- when the moisture content was 40 and
Soluble SO42- in soil is determined mainly by the 90 % of the field capacity, respectively. Limiting levels
turbidimetric method, where sulfate is extracted from of water potential for S oxidation vary according to
the soil with monocalcium phosphate or ammonium the soil type, depending on texture and degree of
acetate, and precipitated with barium chloride. The aeration (Janzen & Bettany, 1987).
turbidity of the suspension is proportional to the
amount of sulfate in the sample, and the absorbance The oxidation of S0 results in H+ generation during
is measured by spectrophotometry at 420 nm the process. However, the degree of acidification varies
(Massoumi & Cornfield, 1963; Vitti, 1988; Cantarella depending on the amount of applied S0 and the soil
& Prochnow, 2001). buffering capacity (Yang et al., 2008). Several studies
Prochnow et al. (1997) described the use of ion have shown an increase in S0 oxidation in alkaline
exchange resin for the extraction of SO42- from soil soils or in response to the addition of CaCO3 (Freney,
samples, with results similar to extraction with 1967; Adamczyk-Winiarska et al., 1975; Nor &
ammonium acetate. The amount of sulfate in soil can Tabatabai, 1977; Czaban & Kobus, 2000; Yang et al.,
also be determined using ion exchange chromatography 2008). According to Adamczyk-Winiarska et al. (1975),
(Ohira & Toda, 2006; Yang et al., 2010). calcium carbonate could improve the conditions for
the development of S-oxidizing microorganisms in the
According to Watkinson & Blair (1993), the soil. Czaban & Kobus (2000) suggested that under
determination of the remaining S0 is more accurate acidic conditions, S0 oxidation is driven predominantly
than the determination of S042- for the evaluation of by soil fungi.
S0 oxidation in soils. The authors argue that during
long incubation periods, the soil organic S may be Vogler & Umbreit (1941) proved the need for a direct
mineralized and/or S0 4 2- can be immobilized, contact between T. thiooxidans, (actually A.
decreasing the efficacy of the methodology. To thiooxidans) and S0 particles for efficient oxidation.
minimize this effect, Janzen & Bettany (1987) The oxidation rate is related to the total area of S0
suggested a shortened incubation period of six days particles, which in turn increases with decreasing S0
prior to sulfate determination. Another drawback of particle size (Freney, 1967). To obtain a greater
determining the remaining S0 is the use of toxic efficiency when S0 is used as fertilizer, the size of the
solvents such as toluene, chloroform and acetone for applied particles must be between 80-1,000 mesh or
S0 extraction from soil samples (Barrow, 1968). In smaller (Wainwright, 1984). The particle shape also
addition, the extraction of S0 with chloroform may be influences the total surface area; the more spherical
incomplete, depending on the levels of soil moisture the shape, the smaller the mass/area ratio (Germinda
or sample drying temperature (Barrow, 1970). Soil & Janzen, 1993). Several mathematical models have
particle aggregates cannot be properly dispersed in been described for the determination of the oxidation
chloroform, difficulting the solubilization of occluded rate depending on the size and shape of S0 particles
S0 and interfering with the accuracy of the analysis over time, and there is an ongoing debate over which
(Watkinson et al., 1987). is the best model (Blair, 1987; Janzen & Bettany, 1987;
McCaskill & Blair, 1987; Blair et al., 1993; Watkinson,
Variables affecting the oxidation of S0 in soil 1993; Watkinson & Blair, 1993).
The main environmental factors influencing S0 As mentioned above, the oxidation rate is inversely
oxidation in soils are temperature, moisture, aeration, proportional to the S0 particle size; however, the
pH, and microbial diversity. Other factors, such as application of very small particles in the field is very
particle size, dispersion in soil and the fertilizer difficult, partly because of the fire risk due to sulfur
formulation also affect the S0 oxidation rate. flammability. Additionally, an inadequate dispersion
In general, the rate of S0 oxidation is minimal in of S0 particles in the soil dramatically decreases the
temperatures below 10 °C and above 40 °C (Freney, oxidation rate (Germida & Janzen, 1993). The same

R. Bras. Ci. Solo, 36:1369-1379

1376 Adriano Reis Lucheta & Marcio Rodrigues Lambais

authors comment that oxidation was limited when S0 ALBUQUERQUE, G.A.S.C.; AZAMBUJA, R.S.L. & LINS,
dispersion in the soil was low (< 50 g soil g-1 S0) and F.A.F. Agrominerais - Enxofre. In: LUZ, A.B. & LINS,
maximal when S0 was diluted to 1,000 g soil g-1 S0. F.A.F., eds. Rochas: Minerais industriais: Uso e
especificações. 2.ed. Rio de Janeiro, Centro de Tecnologia
However, optimal conditions may vary according to
Mineral - CETEM, 2008. p.577-590.
the soil type. The use of S0 in bentonite, covered urea
or mixed with rock phosphate facilitates field ANANDHAM, R.; INDIRAGANDHI, P.; MADHAIYAN, M.;
application. In contrast, an increase in surface area KIM, K.; YIM, W.; SARAVANAN, V.S.; CHUNG, J. & SA,
decreases the oxidation rate in the soil (Boswell & T. Thiosulfate oxidation and mixotrophic growth of
Friesen, 1993; Boswell et al., 1996). Methylobacterium oryzae. Can. J. Microbiol., 53:869-876,
2007.
Finally, a previous S0 application to a particular
area has a positive effect on the S0 oxidation rate ANANDHAM, R.; INDIRAGANDHI, P.; MADHAIYAN, M.;
observed after the second application. Solberg et al. RYU, K.Y.; JEE, H.J. & SA, T.M. Chemolithoautotrophic
(2005) observed that the SO42- recovery was between oxidation of thiosulfate and phylogenetic distribution of
sulfur oxidation gene (soxB) in rizhobacteria isolated from
1.88 to 3.13 times greater after the second S0 application
crop plants. Res. Microbiol., 159:579-589, 2008.
compared to the first. However, according to Li et al.
(2005), in soils that already had a high oxidation rate BAGCHI, A. & ROY, P. Structural insight into SoxC and SoxD
after the first application, a second S0 application did interaction and their role in electron transport process in
not necessarily improve this rate. In contrast, in soils the novel global sulfur cycle in Paracoccus pantotrophus.
with low initial oxidation rates, an increase in oxidation Biochem. Biophys. Res. Comm., 33:1107-13, 2005.
can be expected after the second S0 application. One BALDENSPERGER, J. Use of respirometry to evaluate sulphur
possible explanation for this phenomenon is the oxidation in soils. Soil Biol. Biochem., 8:423-427, 1976.
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BLAIR, G.J.; LEFROY, R.B.; DANA, M. & ANDERSON, G.C.

Modelling of sulfur oxidation from elemental sulfur. Plant
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