Community structure of methanogenic archaea and methane production associated with compost

17 Pages·2012·0.68 MB·English

RESEARCH ARTICLE
Community structure of methanogenic archaea and methane
production associated with compost-treated tropical rice-field
soil
Alpana Singh1, Ram S. Singh2, Siddh N. Upadhyay2, Chaitanya G. Joshi3, Ajay K. Tripathi3 & Suresh
K. Dubey1
1DepartmentofBotany,FacultyofScience,BanarasHinduUniversity,Varanasi,India;2DepartmentofChemicalEngineeringandTechnology,
InstituteofTechnology,BanarasHinduUniversity,Varanasi,India;and3DepartmentofAnimalBiotechnology,AnandAgriculturalUniversity,
D
Anand,India o
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Correspondence:SureshK.Dubey, Abstract h
DepartmentofBotany,FacultyofScience, ttp
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BanarasHinduUniversity,Varanasi221005, The diversity and density of methanogenic archaea and methane production ://a
India.Tel.:[phone];fax: were investigated ex situ at different growth stages of rice plant cultivated in ca
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[phone];e-mail: compost-treated tropical rice fields. The qPCR analysis revealed variation in e
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[email protected] methanogens population from 3.40 9 106 to 1.11 9 107 copies g(cid:1)1 dws, in the ic
year2009and4.37 9 106to1.36 9 107 copies g(cid:1)1 dwsintheyear2010.Apart .ou
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Received16February2012;revised26April from methanogens, a large number of bacterial (9.60 9 109–1.44 9 1010 cop- .c
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2Fi0n1a2l;vearcscioenptpedub8lisMheadyo2n0l1in2e. 13June2012. ies g(cid:1)1 dws) and archaeal (7.13 9 107–3.02 9 108 copies g(cid:1)1 dws) communi- m/fe
ties were also associated with methanogenesis. Methanogen population size m
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DOI:10.1111/j.1574-6941.2012.01411.x varied in the order: flowering > ripening > tillering > postharvest > preplanta- ec
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tion stage. The RFLP-based 16S rRNA gene-targeted phylogenetic analysis rtic
Editor:AlfonsStams showed that clones were closely related to diverse group of methanogens com- le
Y -a
prising members of Methanomicrobiaceae, Methanosarcinaceae, Methanosaeta- b
G Keywords ceae and RC I. Laboratory incubation studies revealed higher amount of stra
O methanogenicarchaea;composttreatment; cumulative CH4 at the flowering stage. The integration of methanogenic ct/8
OL strtorupcictuarles.oil;CH4production;community uconmdemrsutannitdyinsgtruocftuthree adnydnaCmHic4sporfodCuHct4iopnropdoutcetniotinalionfosrogilanriecsaullltyedtreinateadbreitcteer- 2/1/118
field soil. The hypothesis that the stages of plant development influence the /5
6
C methanogenic community structure leading to temporal variation in the CH4 541
E productionhasbeensuccessfullytested. 6
b
y
Y g
u
e
G s
Introduction ecosystems. Environmental concerns and need for the t o
O sustainability of agricultural production have shifted the n 0
4
L The significant increase in the atmospheric level of meth- focus to organic agriculture, which enhances the fertility A
p
O baneceau(sCeHo4f),incorveearsintgheanpthasrtoptowgoeniccenemtuirsiseisoniss, wphriimcharairlye olofgaicgarlo-aecctoivsiytsyte(mFAaOn/dWpHroOm;oFteAsOs,oi1l9b9i9o)d.ivInersIintydiaan,dabboiou-t ril 20
BI currently around 1.5–2.5 times the natural emissions 528 171 hectares (0.3% of the total agricultural land) are 19
O (Forster et al., 2007). The flooded rice fields are the under organic farming (Ramesh et al., 2010). It is
major anthropogenic sources owing to the prevailing expected that there will be substantial expansion in
R
anaerobic conditions. Several factors are known to influ- organic farming-based rice cultivation in the near future.
C
ence CH emissions from rice fields. Field and laboratory Thus, study and analysis of the dynamics of methanogen-
4
I
M studies have indicated the influence of soil types (Yao ic community have become imperative for the assessment
et al., 1999; Conrad et al., 2008), application of chemical of CH production from such rice fields.
4
fertilizers (Hou et al., 2000; Kruger & Frenzel, 2003; Liou In spite of increasing emphasis on organic farming,
et al., 2003), and cultivars (Liou et al., 2003; Jia et al., only a limited number of studies focusing on various
2006) on the dynamics of CH production in rice-field parameters affecting methane production in compost-treated
4
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol82(2012)118–134
PublishedbyBlackwellPublishingLtd.Allrightsreserved
Methanogeniccommunityintropicalricesoil 119
rice fields are available in the open literature. A summary present study examines the methane production
of the reported published information on methane pro- potential of soil, assesses the diversity of methanogenic
duction in compost-treated soil of rice fields together community and enumerates the population size of
with other relevant factors is presented in Table 1. It is methanogens along with bacteria and archaea, at differ-
seen that only Lu et al. (2000), Kumarswamy et al. ent stages of compost-treated rice cultivation including
(2001), Peng et al. (2008) and Supparattanapan et al. preplantation and postharvest stages. The rate of CH
4
(2009) have used soil from tropical rice fields, rest all production has been quantified using an appropriate
have used soil from cold or temperate region rice fields. kinetic model.
Methane production has been studied by all except
Weber et al. (2001), Sugano et al. (2005), and Watanabe
Materials and methods
et al. (2010). Microbial population size has been studied
D
only by Hou et al. (2000), Kumarswamy et al. (2001) ow
Experimental site and soil sampling n
and Conrad & Klose (2006). The available information lo
a
on the methanogenic population size and community The soil was sampled during the cropping seasons of d
e
d
structure during various stages of rice cultivation is also years 2009 and 2010 from the field located at Surbhi fro
limited, although the presence of methanogenic commu- Sodh Sansthan, Dagmagpur, Mirzapur district of Eastern m
nity related to Methanomicrobiaceae, Methanosarcinaceae, UP, India (82°40′30″E, 25°6′30″N) where organic farming http
MI (eRthCanIo)saheatsacbeeaeen, MobestehravneodbabcyteWriaecbeeareeatnadl. R(2i0ce01C),luSsutge-r itsypbeeinhgavpinragctsiacneddfo34r%th,espilatst641%1 yeaanrds.cTlhayis5so%il,ispHAlfi6s.o8l, s://ac
a
ano et al. (2005), Conrad & Klose (2006), Peng et al. and soil organic carbon 0.8%. The region is classified as de
m
(2008) and Watanabe et al. (2010). Except the work of Vindhyan foot-hill in the Indo-Gangetic plane, and the ic
Peng et al. (2008), all other studies are limited to rice climate is relatively dry owing to deficit rain which is .ou
p
fields of cold and temperate climates. Therefore, analysis normally 1100 mm annually. The annual precipitation .c
o
of density and diversity of methanogenic community during the period of study was 502 and 670 mm, respec- m
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and their relation with CH4 production in tropical rice tively. Most of the rainfall is confined during June to m
s
fields, particularly of the Indian sub-continent, is of September in the monsoon season. The minimum tem- e
c
utmost importance, to bridge the knowledge gap. perature varies from 8 °C in January to 29 °C in June, /a
Earlier studies have shown that there is considerable and the maximum ranges between 28 and 42 °C, respec- rticle
variation in CH flux in relation to plant age (Wang & tively. The cropping sequence of rice-wheat-rice is being -a
4 b
s
Adachi, 2000; Jia et al., 2006). As quality and quantity of practiced at the site since long. The experimental site has tra
exudates released from root vary during crop growth, been initially a water-eroded area which has been ct/8
there is a possibility of subsequent change in methano- brought under agricultural practice based on organic 2/1
genic community structure in relation to plant age. In farming concept since 2000. Manure is applied during /1
1
addition, it is also known that a strong plant–microbe summer at the rate of 15 tons ha(cid:1)1. Manure is prepared 8/5
6
interaction is sustained in soil at the cost of mutual bene- from cow dung and crop residues using NADEP tech- 5
4
1
fit. Once the interaction gets disturbed because of crop nique developed by Nana Deshmukh Pandharipande of 6
b
harvest, one can expect alteration in the community Maharashtra (India) for preparing compost. This is a y
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structure owing to the absence of crop and flooding con- popular method for the formation of compost (organic u
e
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dition in the field. Therefore, a correlation between fertilizer) in India (Chandra et al., 2007). The ripe man- t o
methanogenic population density and diversity with ure is dispersed manually by fieldworkers and then n 0
4
methane production potential of soils throughout the homogeneously mixed by ploughing up to 15 cm depth A
p
ethnetirmeiccrroopbpiainlgecpoelroiogdy wofouCldHb4ephreoldpufucltiionnuinnderriscteanfideilndg. tnoutirniecnotrpoarpaptleiciattioinnthise emntaidree peloxcuegpht laaymere.ndNmoefnutrsthoerf ril 201
Further, a clear understanding of the role of methanogen- diluted cow urine (1 : 50) at an interval of 40 days as a 9
ic community in modulating the CH production is supplement of nutrients during crop growth which pro-
4
essential for developing process-based models for CH vides mainly nitrogen. The irrigation was carried out as
4
flux from rice ecosystems as well as for elucidating the and when required. Soil samples in triplicate from the
linkage among phenology, plant activity and soil rhizosphere were collected at different stages of rice crop
microbial community dynamics. cultivation such as preplantation, tillering, flowering, rip-
Our previous study has demonstrated the temporal ening and postharvest using method described in Dubey
change in methanogenic diversity and CH production & Singh (2000). Soil samples were brought to laboratory
4
potential of soil during crop cultivation in a nitrogen and kept at (cid:1)20 and 4 °C for molecular and soil
fertilizer-amended rice field (Singh & Dubey, 2012). The analyses, respectively.
FEMSMicrobiolEcol82(2012)118–134 ª2012FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
120 A.Singhetal.
0) g
References Chidthaisongetal.(1999) etal.Lu(200 etal.Hou(2000) Kumarswamyetal.(2001) Yang&Chan (2001) etal.Weber (2001) etal.Sugano (2005) etal.Yan(2005)
1
(cid:1)
g
9510(cid:1)1g 5MPN
Methanogens populationsize – – 9–44.5102.3methanogens drysoil 9Upto6110drysoil – – – – Download
e
d
post fro
typestreatedwithcom Methanogens community – – – – – RCI,RCIV,Methanosarcinaceae,Methanomicrobiaceae,Methanobacteriaceae RCI,RCVI,Methanomicrobiales,Methanosarcinales,Methanobacteriales,Crenarchaeota– m https://academic.ou
soil p.c
eniccommunityacrossvarious Methaneproduction (cid:1)1Upto200nmolgsoilfor(cid:1)c1CM,.25nmolgsoil forRCandRS (cid:1)l1gUptoabout8molCH4(cid:1)1dwsday (cid:1)–l1204499gCHgsoil4 lUpto25492.9molCH4(cid:1)1kgsoil (cid:1)(cid:1)21hUpto1.42mgm – – emission3.1timesCH4greaterinorganicamended soilfromsoilwithoutanyorganicamendment om/femsec/article-abstract/8
g 2
no /1
alstudiesonCHproductionandmetha4 Methods Fieldtreatmentofcowmanure(CM),ricestrawcompost(RC)+andricestraw(RS).10gsoil10mLdeionizedwater,incubated°at30Cindarkfor30days20gsoilwithricestraw(0.2g)was–°incubatedat2535Cover56days Soiltreatedwithricestrawandmanures(pig,cattle,chicken);20g+soil100mLwaterincubatedfor°40daysat30C.Population countedbyMPNmethod 5gsoilwithsterilewater(1:1.25ratio)wasincubated withricestraw(1%w/w)for30daysinvacutainertubes. PopulationcountbyMPNmethodSesbaniaGreenmanure()treated soilcoreof5cmlengthwasincubatedinserumbottleswith freewaterfor140daysO2++40gdrysoil0.5gstraw 40mLdeionizedwaterin150mLglassbottles.Incubated°for10weeksat25Cindark.Communityanalysesusing cloning-sequencing,T-RFLPRicestrawamendedsoilsamples werecollectedduringricecultivation.Methanogenic archaealcommunitywasanalysedusingPCR-DGGE-Sequencing Fieldtreatmentwithdriedricestraw(cid:1)1beforericeapplicationat6thaplanting /118/565416 by guest on 04 April 201
pic 9
SummaryofselectedtyTable1. Ecozone ShigaPrefectureAgriculturalStation, CentralJapan Mollisol,Inceptisol, EntisolandAlfisolofLagunaProvince,Phillipines Sandysoil,YongfengCounty,Beijing,China Deltaicalluviumsoil,Orissa,India Haplanthreptsandyloamand Putoclayloamsoil,TaipeiCityandTaoyuan,Taiwan Sandyloamsoil,Vercelli,Italy OxyaquicDystrudeptsoil, CentralJapan Asianricefieldsoil
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol82(2012)118–134
PublishedbyBlackwellPublishingLtd.Allrightsreserved
Methanogeniccommunityintropicalricesoil 121
References Conrad&Klose(2006) etal.Peng(2008) Supparattanapanetal.(2009) etal.Watanabe (2010)
Methanogens populationsize Hydrogenotrophs:(cid:1)–961g336710 dws.Acetotrophs:–9613721310(cid:1)1gdws – – – Download
e
d
Methanogens community Methanosarcinaceae,Methanosaetaceae,Methanomicrobiaceae,Methanobacteriales,RCI Methanosarcinaceae,Methanosaetaceae,Methanomicrobiaceae,Methanobacteriaceae,RCI,II,III,IV,V,LDS, Crenarchaeotalgroups– Methanosarcinales,Methanomicrobiales,Methanobacteriales,MethanocellalesandRCI from https://academic.ou
p
.c
o
m
Methaneproduction (cid:1)–1g154157nmolCH4(cid:1)1dwsh productionwasRateofCH4higherinstrawtreatedsoilcomparedtoroottreated (cid:1)2Upto544mgCHm4(cid:1)1dayforricestrawand(cid:1)(cid:1)21529mgCHmday4forcowmanure– /femsec/article-abstract/8
2
/1
Methods 6gsoilincubatedwith6mLOfree2+sterilewater120mgricestrawat°25Cfor40days.Community analysesusingT-RFLPofarchaeal16SrRNAgenes.Populationcountby Real-timePCR+Incubationexperiment:10gsoil+20mLdegassedwaterricestrawandrootresidues(0.1g)incubated°for90daysat15,30or45C.DiversityanalysisbyT-RFLPusing archaeal16SrRNAgeneFieldstudywithricestraw(6.25(cid:1)1)andcowmanureamendmenttha(cid:1)1(8.99tha) Ricesoilsamendedwithricestraw, compostingricestrawandplantresidueswerecollectedfor MethanogenicarchaealcommunityanalysisusingDGGE /118/565416 by guest on 04 April 201
9
c
ContinuedTable1. Ecozone Siltloamsoil,Vercelli,Italy Ricefieldsoil,Hangzhou,China Coarseloamysoil,North-East, Thailand AnthraquicYellowsoil(Oxyaqui Dystrudept),Graylowlandsoil(Endoquepts)andAnthraquic Melanudand(Andisols),Japan
FEMSMicrobiolEcol82(2012)118–134 ª2012FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
122 A.Singhetal.
ATTTCAC-3′) as described by Watanabe et al. (2004).
Soil physicochemical and plant biomass
The reaction mixture contained 2.5 lL of 109 buffer
analyses
(Fermentas, UK), 100 lM each of dNTPs (Fermentas,
The physicochemical properties of soil such as texture, UK), 0.5 lM each of primer (Sigma Aldrich) and 1 U
WHC, organic C, total-N, NHþ-N (American Public Taq DNA polymerase (New England Biolabs, Beverly,
4
Health Association, 1985) and biomass of rice plants MA). Amplification was performed in a total volume of
(root and shoot dry weight; Dubey & Singh, 2000) were 25 lL in 0.2-mL reaction tubes using DNA thermal
determined as per the standard methods. cycler (My CyclerTM Thermal Cycler; Bio Rad Laborato-
ries, Inc., Australia) as per the protocol given by Watana-
be et al. (2004). PCR-generated products were used for
Methane production
cloning (Vishwakarma et al., 2010). Positive insert con-
D
To monitor methane production, anoxic sterile water was taining amplified PCR product of each single clone ow
added to 50 g of soil in 250-mL flasks to provide a (10 lL) was digested in the PCR buffer with 1 U of AluI nlo
a
10-mm standing water layer above the soil surface. The and HaeIII (New England Biolabs, Beverly, MA; Wright d
e
d
flspaesckisallwyedreesisgenaeleddowpeitnhingas,btuwtoylforurbNberflsutsohpinpegranhdavitnhge &tionPimatm3,72°0C0.3)Deigaecshtedanpdrokdeputctsovwerenreighatnaulynsdeedrtihnrcouubgah- from
2 h
third one with a rubber septum for gas sampling. Before electrophoresis (3% agarose gel). ttp
ginacsufboartio3nm, itnhewhitehadcsopnascteanwtasshapkuirnggedofwflitahsksO2tofreenesuNre2 tioTnhaletcalxoonneosmthicusuonbittsain(OedTUwse)reondistthriebubtaesdisinotfodoispteinrac-t s://ac
a
anaerobic environment. The flasks (in triplicate) were RFLP patterns. The restriction patterns thus obtained de
dark-incubated statically at 30 °C for 20 days. Methane were used for generating zero-one matrix and dendro- mic
in the headspace was measured using gas chromatograph gram using NTSYS software (Version 2.2Windows© .ou
p
(GC Varian 3800, The Netherlands) equipped with a 2002; Applied Biostatistic Inc., Australia). At least one .c
o
Flame Ionization Detector and a Porapack Q Column representative clone or two clones from each group were m
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(3 m). The column, injector and detector temperatures sequenced. Using more than one restriction enzyme for m
were maintained at 80, 70 and 200 °C, respectively. The grouping of OTUs reduces the chances of similar restric- se
c
carrier gas (N ) flow rate was set at 30 mL min(cid:1)1. Sam- tion pattern by two different sequences. According to /a
pling of 1 mL2of gas out of the headspace was performed Wright & Pimm (2003), these enzymes give the best rticle
after vigorous shaking of the flask to allow equilibrium result based on the number of cleavage sites and the dis- -a
b
s
between the liquid and gas phases. Methane production tance between fragments length differentiate the methano- tra
potential of each soil sample was calculated from the CH4 gens from each other. On this basis, one or two clones ct/8
concentration in the head space measured at intervals of from a group are good enough for the analysis of 2/1
0, 60, 120, 180, 240, 300, 360, 420 and 480 h. The meth- community structure. Close relatives and phylogenetic /1
1
8
ane production volumes thus measured at different time affiliations of the sequences were determined using the /5
6
intervals for different stages of plant growth were used to BLAST search program available at the National Centre 5
4
1
evaluate the kinetic constants for methane production. for Biotechnological Information (NCBI) website. A phy- 6
b
logenetic tree was constructed by 1000-fold bootstrap y
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analysis using the neighbour-joining method, ClustalW u
DNA extraction and quantification es
program and NJ plot software (Higgins et al., 1994). t o
n
Total genomic DNA was extracted from the rhizospheric 0
® 4
soil samples (0.5 g) by Fast DNA Spin soil kit (M P A
Real-time quantitative PCR (qPCR) p
BDiNomAepdriecpal;sM, LLPCB,ioS)olfoonr,lyOsiHs)ofucseinllgs aasbpeeardthbeeamtearnu(Ffaacs-t Population of methanogens, archaea and bacteria were ril 20
1
turer’s protocol in triplicate, and the DNA was finally quantified using specific primer pairs as given in Table 2. 9
pooled. The DNA concentration was measured using a Real-time quantitative PCR (qPCR) was performed using
Nanodrop spectrophotometer (ND 1000). Applied Biosystems 7500 Fast Real-Time PCR system
containing 96-well plate (ABI 7500). Each 20 lL of the
reaction mixture contained: 10 lL SYBR-Green I Master-
PCR amplification, cloning, RFLP analysis,
mix of Fermentas (109), 1 lL of each primer (10 lM),
sequencing and assignment of clones into OTUs
2 lL of each DNA template and then maintained by Milli
The sets of primers used for PCR amplification of Q. Thermal cycling conditions for methanogens, archaea
16S rRNA gene of methanogens were 0357F (5′-CCCTA and bacteria comprised initial denaturation at 95 °C for
CGGGGCGCAGCAG-3′) and 0691R (5′-GGATTACARG 10 min followed by 45 cycles of denaturation at 95 °C
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol82(2012)118–134
PublishedbyBlackwellPublishingLtd.Allrightsreserved
Methanogeniccommunityintropicalricesoil 123
for 15 s (Luton et al., 2002; Fierer et al., 2005; Yu et al., results of multiple analyses of variance for various param-
2005). Final extension was performed at 72 °C for 2 min eters, such as NHþ-N content, bacteria, archaea, metha-
4
in all cases. At the end of the run, melt-curve analysis nogens population size, CH production and plant
4
followed by gel electrophoresis was performed to discrim- biomass, are summarized in Table 4.
inatebetweenspecific ampliconsand unspecific SYBR-Green
signals (primer dimers). Standard curve was generated by
Ammonium-N content and plant biomass
linear regression obtained between threshold cycle (C)
t
and copy number of target gene with R2 values of 0.987, NHþ-N concentration varied between 3.7 and 6.4 lg g(cid:1)1
4
0.998 and 0.997, for bacteria, archaea and methanogens, soil in the year 2009 and from 3.4 to 6.5 lg g(cid:1)1 soil in
respectively, and used for the estimation of gene abun- 2010 during various stages of rice cultivation. NHþ-N
4
dance in unknown DNA samples. concentration was the lowest during the postharvest stage
D
and highest during the flowering stage in both the years. ow
Statistical analyses MANOVA indicated significant differences in NHþ4-N nloa
concentration during the years and the sampling stages d
e
þ d
Data were subjected to multivariate analysis of variance (Table 4). The HSD test suggested that NH4-N concen- fro
(MANOVA) to determine the effect of year and sampling tration during flowering stage differed significantly. No m
h
stage on the tested parameters. Tukey’s HSD test (at significant difference was found between preplantation, ttp
mPe<an0.0v5a)luewsa.sRaepgrpelsiesidonfoarnaelsytsimisawtinasgutsheeddtioffedreetnecremsinine tillTerhinegshaonodtpaonsdtharorvoetstbsiotamgeass.s varied, respectively, from s://ac
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the relationship of methanogenic population with 10.20 to 21.80 and 1.00 to 2.10 g per plant in 2009 and de
NanHalþ4y-seNs awndereCHp4erpforromduecdtiounsinpgoteantiSaPlSSof1s6o.i0ls.sAtaltlisstuiccahl f2r0o1m0 (1T1a.5b7let3o).2M2A.5N0OVaAndind1i.c1a0tedtosi2g.n4i0ficganpterdifpflearnentciens mic.ou
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package. in root and shoot biomass values for various cultivation .c
o
stages (Table 4). Tukey’s HSD test indicated that values m
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of shoot biomass varied significantly across the vegeta- m
Results
s
tion stage, while for the root biomass, significant e
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Table 3 lists the observed variation of NHþ-N content differences were observed between tillering and flowering /a
and plant biomass for the years 2009 and4 2010. The stages. rticle
-a
b
s
Table2. DescriptionofprimersandthermalconditionsusedinqPCRassay trac
t/8
Annealing 2
/1
Primer temperature Extension Product /1
Targetmicrobe name Primerset (°C) time(s) size(bp) Reference 18
/5
Bacteria Eub338 F:5′ACTCCTACGGGAGGCAGCAG3′ 53 10 200 Fiereretal. 65
4
Eub518 R:5′ATTACCGCGGCTGCTGG3′ (2005) 16
Archaea ARC787 F:5′ATTAGATACCCSBGTAGTCC3′ 60 15 273 Yuetal. b
y
ARC1059 R:5′GCCATGCACCWCCTCGT3′ (2005) g
u
Methanogens mcrA-f 5′GGTGGTGTMGGATTCACACARTAYGCWACAGC3′ 55 30 491 Lutonetal. es
mcrA-r 5′TGTGCAAGGAGCAGGGAC3′ (2002) t o
n
0
4
A
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Table3. VariationsinNHþ4-Ncontentandrootandshootbiomassofplantduringricecultivation ril 2
Samplingstages Year NHþ-N(lgg(cid:1)1) Shootbiomass(gperplant) Rootbiomass(gperplant) 01
4 9
Pre-plantation 2009 4.30±0.26 0 0
2010 4.13±0.20 0 0
Tillering 2009 4.60±0.25 10.20±0.83 1.0±0.23
2010 4.80±0.32 11.60±1.76 1.1±0.15
Flowering 2009 6.40±0.18 18.50±1.00 2.1±0.25
2010 6.50±0.25 20.23±2.40 2.4±0.22
Ripening 2009 5.20±0.21 21.80±1.80 1.7±0.19
2010 5.13±0.24 22.50±1.29 1.9±0.18
Post-harvest 2009 3.70±0.17 0 0
2010 3.43±0.19 0 0
FEMSMicrobiolEcol82(2012)118–134 ª2012FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
124 A.Singhetal.
Table4. Multivariateanalysisofvarianceshowingeffectofsamplingstageandyearonvariousparameters(P<0.05)
2009 2010 Pooled(2009and2010)
Parameters F P F P Year:F P Stage:F P
4,10 4,10 1,20 4,20
NHþ-N 21.6 0.000 21.83 0.000 0.02 0.893 43.02 0.000
4
CH production 113.28 0.000 88.25 0.000 1.66 0.213 198.39 0.000
4
Bacteria 5.15 0.016 4.33 0.027 3.97 0.06 9.04 0.000
Archaea 88.67 0.000 56.03 0.000 31.59 0.000 125.81 0.000
Methanogens 46.19 0.000 67.17 0.000 26.28 0.000 111.64 0.000
Rootbiomass 6.23* 0.034 13.14* 0.006 1.27† 0.282 17.85‡ 0.000
Shootbiomass 382.75* 0.000 519.68* 0.000 30.58† 0.000 875.23‡ 0.000
*F2,10;†F1,12;‡F2,12. Do
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9
Fig.1. Changesinmicrobialpopulation(bacteria,archaeaandmethanogens)acrossdifferentsamplingstagesofcompost-appliedrice-fieldsoil.
Letters a, b, c and d, wherever common, indicate that the microbial populations are not significantly different (P<0.05 by Tukey’s HSD test),
andthebarindicatesextentofvariationfromthemean.
and 2010. It is seen that the average numbers of bacteria,
Microbial population size
archaea and methanogens varied from 9.60 9 109 to
Figure 1 shows the variation in microbial population dur- 1.39 9 1010, 7.13 9 107 to 2.44 9 108 and 3.40 9 106 to
ing different stages of rice cultivation for the years 2009 1.11 9 107 copies g(cid:1)1 dws, respectively in the year 2009,
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol82(2012)118–134
PublishedbyBlackwellPublishingLtd.Allrightsreserved
Methanogeniccommunityintropicalricesoil 125
whereas during 2010, these varied from 1.02 9 1010 to ripening stages, while clones closest to Methanosarcinaceae
1.44 9 1010, 7.94 9 107 to 3.02 9 108 and 4.37 9 106 to were found in all sampling stages except in preplantation
1.36 9 107 copies g(cid:1)1 dws, respectively. stage sample.
MANOVA indicated that the variation in population size On the basis of substrate level, both hydrogenotrophic
with year and sampling stage was significant for archaea and acetoclastic methanogens were found throughout the
and methanogens. Variation in bacterial population was cropping period with varying abundance. Acetoclastic
significant owing to sampling stages and in-significant community was represented by Methanosaetaceae and
during the years (Table 4). According to Tukey’s HSD Methanosarcinaceae which were less abundant in the early
test, flowering stage showed significant variation with all stage but gradually increased during the flowering and
other stages for bacterial population. Archaeal population then decreased during the ripening stage of rice crop and
varied significantly in all other sampling stages except were also detectable in the postharvest period.
D
between preplantation and postharvest, and between til- Only five sequences of cultured members of methano- ow
n
lering and ripening stages. The methanogenic population gens were nearly identical to the sequenced OTUs of this lo
a
size varied significantly between different stages except study while remaining were related to uncultured metha- d
e
d
preplantation and postharvest, and between tillering and nogen sequences (Fig. 2). Blast analysis showed that clone fro
ripening stages. MG2 of the postharvest stage was closely related to m
h
Methanoculleus marisnigri (99%) and clone U2 of the rip- ttp
Diversity of methanogens ening stage was related to Methanosaeta thermophilum s://a
(99%). MT4 of tillering and MG4 of postharvest stages, c
a
One hundred white colonies were recovered for the con- respectively, have shown 95% and 96% similarity with de
m
struction of clone libraries from each stage sample. Of cultured Methanocella paludicola SANAE. Thus, it can be ic
these, two hundred and forty clones with M13-positive inferred that some methanogenic groups become domi- .ou
p
inserts (48 clones from each stage) were screened for nant and some recessive during the five stages of cultiva- .c
o
RFLP. The RFLP analysis revealed 37 unique OTUs: five tion, contributing to the variation of the methanogenic m
/fe
(MG6, MG7, MG8, MG9 and MO3), nine (MT1, MT2, community throughout the cropping period. m
s
MT3, MT4, MT6, MO1, MO2, MO4 and MO5), eleven In addition to methanogens, the genetically related e
c
(S1, S2, S3, S4, S5, S7, S8, S9, S10, MG3 and MT5), seven nonmethanogenic groups belong to RC III, RC IV and /a
(U1, U2, U3, U4, U5, U6 and S6) and five (MG1, MG2, Thermoplasmatales, in which RC IV-related clones are rticle
MG4, MG5 and MT7) for preplantation, tillering, flower- found in all vegetation stage samples, RC III in only flow- -a
b
s
ing, ripening and postharvest stage, respectively. ering stage and Thermoplasmatales in flowering and rip- tra
Phylogenetic tree shows that total observed clones show ening stage sample. The two representative clones, S5 of ct/8
resemblance with six groups of Euryarchaeota: Methano- flowering stage and U5 of ripening stage, showed similar- 2/1
sarcinaceae, Methanosaetaceae, Methanomicrobiaceae, Ther- ity (99%) with uncultured Thermoplasmatales belonging /1
1
8
moplasmatales, RC I and III, and one group of to deepest Phreatic sinkhole (Sahl et al., 2010). The close /5
6
Crenarchaeota, that is, RC IV (Fig. 2). Phylogenetic relationship was found between clone U3 of ripening 5
4
1
analysis indicates that clones obtained from preplantation, stage and uncultured clone of Taihu lake sediment sample 6
b
tillering, flowering, ripening and postharvest stage soil (Ye et al., 2009). The RC III, RC IV and Thermoplasma- y
g
samples show close genetic resemblance with two tales form deep lineage with methanogenic groups. u
e
s
(Methanomicrobiaceae, RC I or Methanocellales), three t o
(Methanomicrobiaceae, Methanosarcinaceae, RC I), four Methane production n 04
(Methanosarcinaceae, Methanosaetaceae, Methanomicrobia- A
p
cMeaeeth,aRnComIi)c,rofboiuarce(aMe,eRthCanIo)saarncdinathcereaee,(MMeetthhaannoosmaeictraocbeiaae-, T25h8e.03mletghaCnHe4pgr(cid:1)o1dduwctsioinn trhaengyeeavra2ri0e0d9,fwrohmerea4s6.i2n0thtoe ril 201
ceae, Methanosarcinaceae, RC I) groups of methanogens, following year, it varied between 51.43 and 9
respectively (Fig. 2). 263.79 lg CH g(cid:1)1 dws for the entire experimental
4
The highest number of methanogenic groups belongs period (Fig. 4). Methane production was minimal in the
to the flowering and ripening stages while the lowest to preplantation stage and maximum in the flowering stage
the preplantation stage. The methanogenic groups were during the period of study. MANOVA indicated significant
found to be distributed among various sampling difference in methane production during sampling stages.
stages with different relative abundance (Fig. 3). The Variation during the years was in-significant (Table 4).
clones resembling with RC I and Methanomicrobiaceae Tukey’s HSD test suggested that methane production in
were detected in each stage. Methanosaetaceae-related the flowering stage differed significantly from all other
clones were specifically found in samples of flowering and stages except the ripening stage.
FEMSMicrobiolEcol82(2012)118–134 ª2012FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
126 A.Singhetal.
91 MO3 I (HM625619)
58 S6 IV (GU564520)
S9 III (GU564523)
Shen-A15(AF399296)
MG6 I (HM569757)
52 Gap-A07(AF399321)
MethanocellapaludicolaSANAE(NR 028164)
55 MT4 II (HM123724)
MG4 V (HM569755) Rice cluster I
Shen-A30(AF399309)
WCHD3-34(AF050620)
96 S2 III (GU564516) (Methanocellales)
MG9 I (HM569760)
96
85 S3 III (GU564517)
MG7 I (HM569758)
57 MG1 V (HM569752)
84 MT3 II (HM123723) D
71 Gap-A13(AF399327) o
MT2 II (HM123722) w
95 RST-6 (AY063601) nlo
74 S8 III (GU564522) a
S10 III (GU564524) de
91 56 73 pTSN1 -I1I9I( MA(GBeUt1h58a62n47o56s18a5)e)tathermophila(M59141) Methanosaetaceae d from
Methanosaetaconcilii(M59146) h
62 93 LT-SUA3- AIV7 6(H(FMJ745358791983)) ttps
61 MMetTh6A I4I1 ((DHQM016283079266)) ://ac
X4Ar30 (AY607255) a
d
Methanosarcinabarkeri(AF028692) e
68 S7 III (GU564521) Methanosarcinaceae m
63 98 50 MT7 V (HMM12T317 2II7 )(HM123721) ic.ou
Methanosarcinaacetivorans(M59137) p
64 Methanosalsumzhilinae(FJ224366) .c
o
91 Methanococcoidessp.(FN547405) m
Methanolobu9s0zindeMriG(E2U V7 1(1H4M135)69753) /fem
95 Methanoculleusmarisnigri(AF028693) s
73 95 MUet2h aInVo (gHenMiu4m38th9e9r2m)ophilum(M59129) ec/a
72 Methan9o6sphaerulaMpGal3u sItIrIi s((HEMU1556690M7050G4))5 V (HM569756) Methanomicrobiaceae rticle-a
MG8 I (HM569759) b
92 SMS-T9-9Pro-1(AB479405)MO1 II (HM625617) stra
64 58 MRR42(AY125717)MO4 II (HM625620) ct/82
LiM 2A-7E(FN646477) /1
85 CandidatusMethanoregulaboonei(EU887821 /1
100 U1 IV (HM438991) 18
52 Amb 16S Garacph- A58350 ((AEFF309291304000)) Rice cluster III /565
99 MT5 III (HM123725) 4
54 ARR19(AJ227932) 16
Gap-A17(AF399330) b
88 100 bManetdh Ta n0o3b5a7cFt/e0r6iu9m1Rs(pA.(BG1U9596895329)5) y gu
60 Methanobrevibactersp.(GQ906576) e
91 MethanothermococcusMspe.t(hAaBn5o1c8a7ld3o1c)occussp.(FJ766848) st on
Haloferaxvolcanii(DQ915833) 0
U5 IV (HM438995) Thermoplasmatales 4
100 Z17MFA83(FJ484288) A
S5 III (GU564519) 86 92 S4 III GU5M64O521 8II (HM625618) pril 20
MO5 II (HM625621) 19
51 LL ADT 32(AM503288)
100 AS01-06(AF225608) Rice cluster IV
80 U4 1V (HM438994)
U6 IV (HM438996)
73 Gap-A04 (AF399319)
82 AS08-25(AF225655)
0.05
Fig.2. Phylogenetic relationship of representative methanogenic 16S rRNA gene clone sequence for rhizospheric soil DNA in different rice
cultivation stages: preplantation (I), tillering (II), flowering (III), ripening (IV) and postharvest (V). Uncultured and cultured NCBI databases are
showingrelationshipwithrepresentativeclones.Thescalebarsrepresentanestimatedof5%sequencedivergence.Bootstrapvaluesabove50%
areshownateachbranchpoint.GenBankaccessionnumbersareindicatedforeachsequencedcloneinparentheses.
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol82(2012)118–134
PublishedbyBlackwellPublishingLtd.Allrightsreserved
Methanogeniccommunityintropicalricesoil 127
D
o
w
n
lo
a
d
e
d
fro
m
Fig.3. Relative abundance of methanogenic communities for different h
samplingstages. ttp
s
://a
c
a
d
e
Kinetics of methane production m
ic
According to Monod kinetics, the rate of formation of .ou
p
methane can be expressed as .c
o
(cid:1) (cid:3) m
ddMt ¼lKmasxþðXðÞMðMÞÞ (1) pFilagn.t4a.gVeairniacltuiodninginprCeHp4lanptraotdiounctaionndppoosttehnatriavlesotfstsaogielsi.n relation to /femse
c
/a
UstanndteranthdeKgsiv≫en(Msi)tu,ahteinonce(M) is small, (X) is nearly con- bspeostndfiitnglivnaelsueassofinKdiacnatdedR2inarethtaebseulafitgeudreins.TTabhlee 5co.rre- rticle-a
(cid:1) (cid:3) b
s
dM ¼lmaxðXÞðMÞ (2) trac
dt Ks Discussion t/8
2
/1
(cid:1)ordM(cid:3) Pmoeptuhlaantioognensisze of bacteria, archaea and /118/56
¼KðMÞ (3) 54
dt Population size and distribution pattern of methanogens 16
b
along with bacteria and archaea across different sampling y
where stages in rice field have been quantified over the entire gu
(cid:1) (cid:3) es
K ¼ lmaKxðXÞ (4) sdeeatseormninofe trhiceecpomlanptosgitreoweftfhectfoorntwmoethsuancceepssriovdeuycetiaorns.to t on 0
s 4
The present study has indicated that the bacterial A
Osion(cid:1)nintegratio(cid:3)n Eqn (3) gives the first-order rate expres- pgar(cid:1)ocp1huadleawatislo)narpeo(9pe.xu6p0laet9citoen1d09t–o1(7.c4.o14n39tr9ib1u011t0e07–ctoo3p.0tihe2se9gm(cid:1)11e0tdh8wans)ocgoepanineed-s pril 2019
ln Mm ¼Kt (5) sis indirectly. Substrates like H2/CO2, acetate and metha-
M (cid:1)M nol, are essential for methanogenesis. The availability of
m
these substrates depends on the presence of bacterial
where M is methane produced at t = t and M is the community other than methanogens such as hydrolytic
m
maximum amount o(cid:4)f metha(cid:5)ne produced. and fermenting, H -producing and homoacetogenic bac-
2
Thus, a plot of ln Mm vs. t will give a straight line teria which are involved in the decomposition of complex
Mm(cid:1)M
with slope eq(cid:4)ual to(cid:5)K, the pseudo-first-order rate con- organic materials (Conrad, 2007). Population size of
stant. The ln Mm vs. t plots for the years 2009 and methanogens varied from 3.40 9 106 to 1.36 9 107
2010 are givenMimn(cid:1)MFig. 5. Least squares analyses gave the copies g(cid:1)1 dws during the entire experimental period of
FEMSMicrobiolEcol82(2012)118–134 ª2012FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved