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ORIGINAL ARTICLE
Molecular identification and in vitro screening of
antagonisticbacteria from agricultural byproduct compost: Effectof
compost on development and photosynthetic efficiencyof tomato
plant
Piyush Chandna & Saaraj Gupta &Manchikatla Venkat Rajam
& Ramesh Chander Kuhad
Received: 11 February 2013 /Accepted: 29 June 2013 /Published
online: 28 August 2013# Springer-Verlag Berlin Heidelberg and the
University of Milan 2013
Abstract The dynamics of mesophilic and thermophilic bac-terial
population of compost was studied. The bacteria popula-tion in the
compost ranged from 109 to 105 CFU g−1 and wasfound to be maximum
during mesophilic phase, and thendecreased during the thermophilic,
the cooling and maturationphases. Assessment of culturable bacteria
by 16S rDNA re-vealed phylogenetic lineage of different polymorphic
classbacilli,γ,β-proteobacteria and actinobacteria. Bacterial
isolatesproduced extracellular enzymes: proteases, cellulase,
xylanase,pectinase, tannase and amylase. Among them, mesophilic
bac-teria exhibited xylanolytic (81.25 %) and cellulolytic (63
%)activity. Thermophilic bacteria showed cellulolytic (75 %)
andxylanolytic (66.6 %) activity, but a few isolates also
producedtannase and pectinase. All bacterial isolates were
observedto cause inhibition of three isolates of Bacillus pumilus
andone isolate each of Staphylococcus sciuri and Kocuria sp.
Thephysiological effect of compost on shoot length, leaf size
andfruit maturation of tomato have been evaluated; the compost(75
g/pot) improved these parameters as compared to knowncompost (SOM).
The efficacy of compost and SOM on photo-chemistry of tomato leaves
was studied, based on imaging-PAM of the chlorophyll fluorescence
parameters. Fv/Fm andelectron transport rate (ETR) were increased
significantly incompost (75 g) amended pot within 30 days of
growth.Likewise, highest Y (II) of photosystem II (PS II) yield
was
found in compost (75 g) pot in 15 days. The findings of
thisstudy proved that the compost comprising of various
bacteriainvolved in degradation of substrates was found to be
beneficialfor enhancement of tomato growth and development.
Keywords Composting . Culturable bacteria .
Molecularidentification . Extracellular enzymes . In vitro activity
.
Physiological response and photosynthetic activity on
tomatoplant
Introduction
Composting is an aerobic biological process, during which
anorganic waste is decomposed into a stable useful product.However,
the end product should be free from pathogens, so thatit can be
used to improve soil quality and fertility. Composting isan intense
microbiological process; little is known about micro-organisms
involved and their activities during specific phases ofthe
composting process (Rebollido et al. 2008). Polymerasechain
reaction (PCR)-based analysis of 16S rRNA genes is apowerful and
essential tool for the study of bacterial diversity,community
structure, evolution and taxonomy (Hongoh et al.2003). In addition,
inferring evolutionary relationships amongvarious divergent groups
is a daunting task.
Polysaccharide-degrading enzymes are widespread in natureand
microorganisms that are obtained from environment sam-ple are
usually the most convenient for their production. For thescreening
of large numbers of bacteria, efficient plate screeningmethods are
a prerequisite. The efficiency of plate countingmay be increased by
a careful selection of medium for a specificactivity. Therefore,
specific and sensitive plate assay techniquesneed to be developed,
suitable either for identifying variousenzyme producing bacteria or
for quantifying these activities inculture supernatants. It has
been reported that the screening of
Electronic supplementary material The online version of this
article(doi:10.1007/s13213-013-0690-1) contains supplementary
material,which is available to authorized users.
P. Chandna :R. C. Kuhad (*)Department of Microbiology,
University of Delhi South Campus,New Delhi 110 021, Indiae-mail:
[email protected]
S. Gupta :M. V. RajamDepartment of Genetics, University of Delhi
South Campus,New Delhi 110 021, India
Ann Microbiol (2014) 64:571–580DOI 10.1007/s13213-013-0690-1
http://dx.doi.org/10.1007/s13213-013-0690-1
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microorganisms with a set of degrading activities or with
aspecific combination of degrading activities is labor-intensiveand
time-consuming (Ten et al. 2004).
Diverse niches exist in the general environment, such assoil,
aquatic, air and compost habitats for unicellular ormulticellular
life forms. Gonzalez et al. (2011) reported thatmicrobial species
exist in perpetual competition with oneanother for suitable
ecological niches to support their surviv-al and growth. The
synthesis of compounds that may kill orlimit the growth of
competing strains or species can promoteniche monopolization. The
released compounds include an-tibiotics, antimicrobial peptides or
low molecular mass toxicmolecules, each of these coupled to the
mechanisms forintrinsic resistance/immunity by the producing
strain(Gonzalez et al. 2010). Several antimicrobial peptides
areproduced by Bacillus subtilis (Babasaki et al. 1985) with
arelatively narrow range of activity against closely
relatedorganisms (Jack et al. 1995), which may kill a narrow
spec-trum of bacteria as compared to other traditional
antibiotics.
To study the effect of the compost on vegetable production,we
used the tomato plant (Solanum lycopersicum, cv PusaRuby). Tomato
is one of the most widely grown vegetable foodcrops in the world,
second only to potato (Nelson 2008). Theeffects of different
cultivation practices or varying environmen-tal conditions on fruit
development and photosynthesis havebeen reported (Hetherington et
al. 1998). Measuring of chloro-phyll fluorescence provides
information on qualitative andquantitative changes in
photosynthesis (Šlapakauskas andRuzgas 2005), which is an indicator
of primary productivityand also the photosynthetic rate
measurements of tomato plant(Roháček et al. 2008). A ratio of
variable to maximal fluores-cence (Fv/Fm) can then be calculated
which approximates thepotential quantum yield of photosystem (PS)
II (Bilger et al.1995), used for the calculation of linear electron
transfer rate(ETR) according to Krall and Edwards (1992) method
andQuantum yield (Y) (Šlapakauskas and Ruzgas 2005).
The present study was undertaken to identify the
bacteriaisolated from compost by 16S rRNA gene sequence. Thevarious
bacterial hydrolase enzymes present in differentphases of compost
were qualitatively determined by usingsubstrate specific plate
assays. Further, in vitro screening anddetection of antagonistic
bacteria was performed by agardiffusion assays. The effect of
compost on growth and de-velopment as well as photosynthesis
efficiency of tomatoplants was also studied.
Materials and methods
Isolation and enumeration of bacteria during composting
The isolation and enumeration of bacteria during compostingwas
done by a method described earlier (Chandna et al.
2013). In brief, raw materials used for agriculturalbyproducts
compost were rice bran (15 kg), wheat bran(10 kg) and rice husk (10
kg). Other additives like grassand leaves (5 kg each) and the
bulking agent ash (2.5 kg)were also used. Nitrogen (N) was enriched
by adding cowdung (25 kg), mustard oil cake (10 kg), cow urine (40
l) andmolasses (4 l). To eliminate the pH variation,
approximately0.6 % (w w−1) of calcium oxide was added to the
compostraw materials during mixing.
During the composting process, the temperature in thepile (5–30
cm from the top) was recorded between 08:00and 10:00 a.m. on a
daily basis using a mercury thermometer.The pile was turned
manually on 15th day of composting,and thereafter every 10th day.
The pile was divided equallyinto quarters before mixing. First, two
opposite quarters weremixed properly, and then the next two. The
procedure wasrepeated four to five times, however, no extra
aeration wassupplied. The samples were collected at every tenth day
byautoclaved forceps in autoclaved zipper bags for
microbialanalysis. The composting was terminated after 50 days,when
the temperature stabilized at 27 °C (near to ambient)and the
finished product was air dried for 10 days, sievedwith a 10 mm
stitch sieve, and then used for analysis.Compost samples were
collected from different temperaturephases (mesophile: 30 and 35
°C, thermophile: 40 and 50 °C,and the cooling and maturation: 30
°C) of compost.
The compost suspensions were prepared by addition of1 g (wet
weight) of compost sample to 9 ml of sterilizedwater and the colony
counting was done as per a proceduredescribed by Chandna et al.
(2013).
Phylogenetic analysis
The phylogenetic relationship of 33 bacterial isolates ofcompost
was reported earlier by our group (Chandna et al.2013). However,
phylogenetic tree was reconstructed bymeans of neighbor-joining
method using the updated versionof MEGA-5 programme (Tamura et al.
2011).
Enumeration and determination of culturable bacterialhydrolase
enzyme activity by substrate-specific platecounting techniques
Culturable bacteria, such as proteolytic, cellulolytic,
xylan-loytic, pectolytic, tannolytic and amylolytic were
enumerat-ed by the substrate-specific plate dilution method. An
initialsuspension was made following the procedure as
describedabove. Aliquots (100 μl) of last dilution were spread
ontomedium containing agar in petri plates with three
replicates.All results were expressed in CFU g−1 dry weight
(afterdrying the samples at 100±5 °C overnight). The media usedfor
qualitative determination of bacterial hydrolase enzymeactivity are
described below:
572 Ann Microbiol (2014) 64:571–580
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(a) Protease: 2 g of agar and 0.1 g of NaN3 were dissolvedin 0.1
M phosphate buffer (pH 7.0±0.5) by heating, andthen 10 ml of 1 %
casein (Sigma-Aldrich, USA) and1 ml of 1 M CaCl2 in 5 mM NaOH
autoclaved (at10 psi) separately were added to the mixture with
basalmedium (NA). After cooling to approximately 40 °C,20 ml of the
mixture was poured into sterilized petridishes (90 mm diameter),
and was allowed to solidify.
(b) Cellulase: 0.8 g of carboxymethylcellulose (sodiumsalt)
(Sigma-Aldrich, USA) and 2 g of agar weredissolved in 20 ml of 0.1
M phosphate buffer(pH 7.0±0.5) by heating, and then autoclaved at10
psi separately were added to the mixture with80 ml of sterilized
basal medium (Nutrient broth).After cooling to approximately 40 °C,
20 ml of mixturewas poured into sterilized petri dishes (90 mm
diame-ter), and was allowed to solidify.
(c) Xylanase: 0.5 g of birch wood xylan (Sigma-Aldrich,USA) and
2 g of agar were dissolved in 20 ml of 0.1 Mphosphate buffer (pH
7.0±0.5) by heating, and thenautoclaved at 10 psi separately, and
were added to80 ml of sterilized basal medium (nutrient
broth).Tests plates were also prepared similarly.
(d) α-Amylase: 0.8 g of soluble starch (Sigma-Aldrich,USA) and 2
g of agar were dissolved in 20 ml of0.1 M phosphate buffer (pH
7.0±0.5) by heating, andthen autoclaved at 10 psi separately, and
were added to80 ml of sterilized basal medium (Nutrient broth).
Testsplates were also prepared similarly.
(e) Tannase: 0.8 g of tannic acid (Sigma-Aldrich, USA) and2 g of
agar were dissolved in 20 ml of 0.1 M phosphatebuffer (pH 7.0±0.5)
by heating, and then autoclaved at10 psi separately, and were added
to 80 ml of sterilizedbasal medium (Nutrient broth). Tests plates
were alsoprepared similarly.
(f) Pectinase: 0.8 g of casein (Sigma-Aldrich, USA) and 2 gof
agar were dissolved in 20 ml of 0.1 M phosphatebuffer (pH 7.0±0.5)
by heating, and then autoclaved at10 psi separately, and were added
to 80 ml of sterilizedbasal medium (Nutrient broth). Tests plates
were alsoprepared similarly.
For screening of all the 33 bacteria isolated frommesophilic (30
and 35 °C), thermophilic (40 and 50 °C) andthe cooling and
maturation (30 °C) phases were grown for18 h at their respective
temperature. Sample wells with 18mmdiameter were made on test
plates using sterilized cork-borer.An aliquot (50 μl) (inoculated
broth) of a particular culturablebacteria isolate (approx 106 CFU
ml−1) was applied to eachwell, and incubated at their respective
temperature for 18 h forthe determination of proteolytic,
cellulolytic, xylanolytic,pectinolytic, tannolytic and amylolytic
properties of the bac-terial isolate. After incubation, proteolyses
and pectinases
were observed as a clear halo around the samples well bystaining
with 5 % trichloroacetic acid and 1 M NaCl respec-tively. Amylases,
tannases, cellulases and xylanases werevisualized as a halo zone by
staining of agar plate.Visualization of amylolysis was carried out
by adding 5 mlof 0.2 % KI and 0.02 % I2 solution. Visualization of
tannasewere carried out by exposing the agar plate to 5 ml of 0.1
MNa2CO3 for 3 min, for cellulases and xylanases were carriedout by
exposing the agar plate to 5 ml of 0.1 % congo red for20 min, and
followed by addition of 1 M NaCl with moderateshaking for 3
min.
In vitro screening
Isolates were inoculated into 50 ml sterilized nutrient brothand
incubated at their appropriate temperature up to opticaldensity
(OD600 of 0.4–0.5), then the cultural broth werefiltered through
Whatman filter paper no. 44 and againrefiltered through a Seitz
filter (G4) by vacuum pressure toobtain cell-free culture
filtrates. The screening wasperformed with the 33 bacterial
isolated from compost. Thein vitro antagonistic activity of all
bacterial isolates wasperformed and they were members of the class
bacilli,actinobacteria and proteobacteria (β and γ). The
antagonis-tic activity of the isolated 20 Bacillus strains,
threeStaphyloccocus strains and one strain each of
Kocuria,Microbacterium, Comamonas, Acidovorax,
Enterobacter,Serratia, Klebsiella was determined using the
indicatorstrain. The fresh bacterial cultures, except Durck17,
weregrown at 35 °C to mid-late log phase with optical density(OD600
of 0.4–0.5) and were used for agar well diffusionassay plate. The
wells cut out (18.0 mm dia) on such plates(two on each plate) were
filled (100 μl) with cell-free bacte-rial culture filtrates of two
different isolates were takenaccording to their temperature. Clear
zones of inhibitionwere produced after overnight incubation of
plates, whichwas used as an indication of the growth inhibition. A
directcomparison was made between the diameters of the zones
ofinhibition produced by different isolates.
Plant material
The tomato (Solanum lycopersicum, cv Pusa Ruby) seedswere sown
in plastic pots (length 4 inc×dia 4 in.) and seedlingswere grown
under controlled growth conditions (26±2 °C,16 h photoperiod with
irradiance of 40 μmol m−2 s−1). Atthe two-leaf stage, tomato
seedlings were transplanted intodifferent pots (length 10 inc×dia 4
in.) that contained 1:1mixture of sterilized soil (without any
added fertilizer) andvermiculite (100 g each), which is
supplemented with differentquantity of commercially purchased
Simbhaoli OrganicManure (SOM-obtained from the Simbhaoli Sugar Mill
Ltd.,Ghaziabad) or compost generated in the present study
Ann Microbiol (2014) 64:571–580 573
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(supplementary Table S1). Pots were kept in the controlledgrowth
conditions as mentioned above in a complete random-ized design.
Tomato plants were watered with tap water whenneeded. For each
treatment, 12 seedlings were maintained andthe experiment was
repeated thrice.
Effect of compost on shoot length, leaf size, fruitingand
maturation
The effect of type of substrate (compost and SOM) and thedose of
substrate (25, 50, 75, and 100 g) were analyzed. Thechanges in each
of the measured parameters as compared toSOM, indicating the plant
responses to the different amend-ments are represented here: the
shoot length and size ofleaves were measured. Additionally,
fruiting and maturationwere also determined for each of the tomato
plants with orwithout compost treatment.
Photosynthetic parameters
Measurements were taken on every 15 days up to 2 monthswith a
pulse-amplitude-modulated photosynthesis yield an-alyzer (Portable
Junior-PAM; Walz, Effeltrich, Germany)under photosynthetic steady
state conditions using a photonflux density of 1,550 μmol m−2 s−1
as actinic light and10,000 μmol m−2 s−1 as saturation flashes (with
a durationof 0.8 s). Junior-PAM fluorometer system contains
WINCONTROL software (Walz, Germany) (Kooten and Snel1990). Changes
in the effective quantum yield of PS II withinitial fluorescence
(Fv), maximal fluorescence (Fm) andpotential quantum yield of PS II
(Fv/Fm) were measured asper method described by Kitajima and Butler
(1975) in fivefully expanded leaves, considering the fully open
leaves. Therelative electron transport rate (ETR), is the product
of theeffective photochemical yield of PS (II). ETR was deter-mined
following the methodology of Genty et al. (1989).Moreover, the
effective photochemical quantum yield {Y(II)} of PS II was also
calculated according to the methoddescribed by Kramer et al.
(2004). These experiments wererepeated thrice, with varying amounts
of compost and SOM(Table 1).
Statistical analysis
The experiment on the qualitative analysis of organic
matterdecomposing bacteria, and the effect of compost on
tomatodevelopment and photosynthetic efficiency were repeated
atleast three times with twelve replicates in each experiment.Data
presented are the average (mean) with standarderror/standard
deviation from all the experiments.
Results
Viable bacteria count and qualitative determinationof bacterial
hydrolytic enzyme activity of compostat different temperatures
Colony count analysis was performed by cultivation-basedmethods
to reveal the changes in the number of bacteriaduring the
composting process as has been described in detailearlier by
Chandna et al. 2013.
The population of proteolytic, cellulolytic,
xylanolytic,pectolytic, tannolytic and amylolytic bacteria were
responsi-ble for the degradation of protein, cellulose, xylan,
starch,pectin and tannic acid respectively (Table 2). The
totalxylanolytic bacterial colonies count were found to be1.3×105
CFU g−1 of compost suspension for mesophilicphase, whereas, for
thermophilic phase, cooling and matura-tion phase, the bacterial
count were progressively decreasedto 104 and 103 CFU g−1,
respectively. Similarly, the counts ofmesophilic-proteolytic,
cellulolytic and amylolytic bacteriawere 100-fold higher with their
respective thermophiles,which were even tenfold higher than cooling
and maturationphase bacteria.
The qualitative screening of bacterial isolates revealedthat 22
isolates degraded xylan (xylanase); 19 isolates de-graded cellulose
(cellulase); 12 isolates degraded protein(protease); 12 isolates
degraded starch (amylase); 5 isolatesdegraded pectin (pectinase)
and tannic acid (tannase) wasdegraded by only 3 isolates in overall
composting (Table 3).High availability enzymatic profiling by
mesophilic bacteriafollowed by thermophilic bacteria and then
bacteria presentat cooling and maturation phase respectively,
utilized thesoluble and readily degradable substrates during the
differentphases.
Molecular identification of the bacterial isolates
The partial 16S rRNA gene sequences (Chandna et al.2013) of
representative isolates were used to reconstructa phylogenetic tree
(Fig. 1) using upgraded version ofMEGA 5.
In vitro screening of antagonistic activity
In vitro screening on NA medium depicted that five of
33bacterial isolates had an effective response in
antagonisticactivity against other bacterial isolates isolated from
com-post. Bacillus pumilus Durck8 inhibited Staphylococcussciuri
Durck1 with a clear zone of inhibition (5.0 mm).Likewise, similar
inhibitory results were recorded for otherisolates (Table 4).
574 Ann Microbiol (2014) 64:571–580
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Effect of compost and SOM on shoot length, leaf sizeand fruiting
maturation on tomato plant
The application of compost with varied concentration like 25,50,
75 and 100 g in each pot of tomato plant promoted the shootlength
and leaf size as compared to similar proportion of SOMsupplemented
pots and nontreated control plants (Table 5).Fruiting maturation
showed marginal improvement in plantssupplemented with compost or
SOM in pots. Early maturationof fruits from mature green stage to
breaker stage happened in2 days with the plants supplemented with
the compost (75 g),whereas SOM-supplemented pots required a
week.
Complementary changes in Fv/Fm, ETR and Y (II) on tomatoplants
leaves
Various aspects of photosystem II were differentially affectedby
compost and SOM substitution for tomato plant leaves, asshown by
the various analyses. The changes in the maximumphotochemical
quantum yield (Fv/Fm), higher electron trans-port rate (ETR) and
effective photochemical quantum yields{Y (II)} at the growth of
compost and SOM-amended potsduring the 60-day bioassay in tomato
leaves were studied.Maximum photochemical quantum yield of
photosystem II(Fv/Fm) showed significantly affected in tomato
plants grown
Table 1 Composition for each of the four different
treatments
Treatment Sterilizedsoil* (g)
Sterilizedvermiculite* (g)
Compost (g) Mixing ratio ofcompost in pot
Labeled ascompost (C)
OrganicSOM (g)
Mixing ratio ofSOM in pot
Labeled asSOM (F)
Treatment 1 100 100 25 200:25 (8:1) C1 25 200:25 (8:1) F1
Treatment 2 100 100 50 200:50 (4:1) C2 50 200:50 (4:1) F2
Treatment 3 100 100 75 200:75 (2.66:1) C3 75 200:75 (2.66:1)
F3
Treatment 4 100 100 100 200:100 (2:1) C4 100 200:100 (2:1)
F4
Fig. 1 Neighbour-joiningphylogenetic tree indicating theposition
of bacteria among therelated species of the genusStaphylococcus,
Bacillus,Terribacillus, Lysinibacillus,Serratia,
Klebsiella,Enterobacter, Microbacterium,Kocuria, Acidovorax
andComamonas using MEGA 5software
Ann Microbiol (2014) 64:571–580 575
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in the presence of compost. Significant increase of Fv/Fm in
allpots of varied compost concentrations like 25, 50, 75 g and
100 g were observed for 30 days, as the days progressive from45
to 60 days, the value of Fv/Fm started to decrease (Fig. 2a),
Table 2 Organic matter-decomposing bacteria in agricultural
byproducts compost
Number of bacterial colonies (CFU g−1 of dry sample)Phase
Qualitative analysis of various bacterial hydrolytic enzymes
Proteolytic Cellulolytic Xylanloytic Pectolytic Tanninolytic
Amylolytic
Mesophilic 9.0×104±0.40 1.06×105±0.03 1.3×105±0.09 2.2×101±0.11
1.8×101±0.10 9.8×104±0.10
Thermophilic 6.7×102±0.15 7.2×103±0.20 8.5×104±0.13 5.2×101±0.2
2.4×101±0.15 3.0×102±0.31
Cooling and Maturation 6.31×101±0.12 2.8×101±0.08 7.1×103±0.2 –
– 7.7×101±0.25
Data is the mean ± standard error (SE), based on three
independent experiments with three replicates in each
experiment
Table 3 Various bacteria show dynamic enzymatic effect in
studied compost
Bacteria Hydrolase’s Temperature & Phase
Protease Cellulases Xylanases Pectinases Tannase Amylase 30 °C;
Mesophilic
Staphylococcus sciuri Durck1 AM778178 − ++ +++++ + − −Bacillus
pumilus Durck14 AM778191 − ++ + − − −
Bacillus subtilis Durck10 AM778185 + ++++ + − + +
Bacillus subtilis Durck7 AM778184 + ++ − − − +
Staphylococcus sciuri Durck9 AM778188 − − − − − −
Bacillus subtilis Durck12 AM778189 + ++++ + − + +
Klebsiella pneumoniae Durck21 AM884577 − − − − − −
Bacillus pumilus Durck8 AM778187 + − + +++ − +
Bacillus flexus Durck15 AM778192 − +++ +++++ − − −
Bacillus flexus Durck6 AM778183 − − − − − −
Serratia marcescens Durck 24 FR865468 + +++++ +++++ − − +
Staphylococcus sciuri Durck16 AM884572 − − + − − −
Microbacterium sp. Durck18 AM884574 − +++ +++ − − − 35 °C;
MesophilicBacillus flexus Durck23 AM884579 − − ++ +++ − −
Enterobacter sakazaki Durck19AM884575 − − − − − −
Bacillus cereus Durck 30 FR865474 − ++ + − − −
Lysinibacillus fusiformis Durck2 AM778179 − ++ ++++ + − − 40 °C;
ThermophilicKocuria sp. Durck22 AM884578 + − + − − +++
Terribacillus halophilus Durck 28 FR865472 − ++++ +++++ + −
−
Bacillus flexus Durck5 AM778182 − ++++ ++ − − −
Bacillus nealsonii Durck 26 FR865470 − +++ ++++ − − −
Acidovorax sp. Durck 31 FR86547 − + ++++ − − −
Comamonas kerstersii Durck 29 FR865473 − + ++ − − − 45 °C;
ThermophilicBacillus benzoevorans Durck 27 FR865471 + − − − − +
Bacillus subtilis Durck17 AM884573 + +++ − − − +
Bacillus pumilus Durck13 AM778190 + + − − − + 50 °C;
ThermophilicBacillus pumilus Durck3 AM778180 − − − − + −
Bacillus subtilis Durck11 AM778186 + + ++ − − +
Bacillus subtilis Durck4 AM778181 +++ − ++ − − ++++ 35 °C;
Cooling and MaturationBacillus sp. RC1 Data not shown
Bacillus sp. RC2 Data not shown
Bacillus licheniformis Durck20 AM884576 − − ++ − − −
Bacillus circulans Durck 25 FR865469 − + +++ − − −
Scale: +++++ ≥ 4–5 cm; ++++ ≥ 3–4 cm; +++ ≥ 2–3 cm; ++ ≥ 1–2 cm;
+ ≥ 0.5 cm
576 Ann Microbiol (2014) 64:571–580
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Fig. 2 a Changes in themaximum efficiency of PS IIphotochemistry
after 15 min darkadaptation of photochemicalquantum yield (Fv/Fm).
Data isthe mean ± standard deviation(SD), based on three
independentexperiments with three replicatesin each experiments. b
Changesin the maximum efficiency of PSII photochemistry of
higherelectron transport rate (ETR).Data is the mean ±
standarddeviation (SD), based on threeindependent experiments
withthree replicates in eachexperiments. c Changes in themaximum
efficiency of PS IIphotochemistry of effectivephotochemical quantum
yieldY(II). Data is the mean ±standard deviation (SD), basedon
three independentexperiments with three replicatesin each
experiments
Ann Microbiol (2014) 64:571–580 577
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whereas, in the 100-g pot the value was slightly less than in
the75-g amended pot. In contrast, significant reductions of Fv/Fmin
all SOM concentration amended pots were observed fortomato plants
(Fig. 2a).
ETR was also affected by compost-amended pots of var-ied
concentration like 25, 50, 75 and 100 g, within 15 dayafter the
start of the treatment; there was only a slight in-crease of ETR
yield in tomato leaves; no major changes inthe ETR of tomato leaves
were observed in 30 day, butsubsequently ETR started to decrease
slightly after 31 day(Fig. 2b). In the SOM-amended pots, similar
trends in termsof days was observed but not in values, as
illustrated inFig. 2b. An early increase in effective photochemical
quan-tum yield Y (II) content in tomato plant leaves was observedin
the 75-g compost amended pot in just 15 days (Fig. 2c). Astime
progressed, the value started declining as depicted in thepreceding
figures, however, under similar conditions a sim-ilar trend was
found in SOM amended pots.
Discussion
Agricultural byproducts produced a good quality of darkbrown
compost depending on the bacterial communitiespropagating in it.
Still, isolation was a necessary approachto obtain bacteria and to
know their physiological character-istics for understanding their
ecophysiological and
environmental functions, and for their application
potentials(Sfanos et al. 2005). 16S rRNA proved to be one of the
mostpowerful tools for the classification of microorganisms(Hanage
et al. 2005) and had been used for the identificationof naturally
occurring bacteria dwelling in compost. A phy-logenetic framework
was constructed using bacterial se-quences and analysis confirmed
the predominance of bacilli,γ-proteobacteria, β-proteobacteria and
actinobacteria (Fig. 1)(Chandna et al. 2013).
Microbial extracellular hydrolytic enzymes were the
majorbiological mechanism for the decomposition of
sedimentaryparticular organic carbon and nitrogen (Dang et al.
2009). Thepresent study revealed that diverse and abundant bacteria
iso-lates secreted at least one or more of the extracellular
enzymeswere screened by using the substrate hydrolysis index
criteriaon the direct cultivation of the isolates on nutrient agar
mediumsupplemented with insoluble substrates. As summarized inTable
2, the mesophilic bacteria especially at 30 °C weredominating and
analyzed through higher CFU g−1 count, whichutilized soluble and
readily degradable substrates.
Bacillus flexusDurck15 and Staphylococcus sciuriDurck1were the
two mesophilic bacteria that produced maximumxylanase. Serratia
marcescens Durck24 and Microbacteriumsp. Durck18 provided maximum
production of both cellulo-lytic and xylanolytic enzyme activity at
30 and 35 °C, respec-tively. The other bacterial isolates were also
investigated onbirch wood xylan, starch, tannic acid and casein and
cellulose-rich mediums and found that xylanolytic activity
was(81.25 %) followed by cellulolytic (63 %) and then amylolyt-ic,
proteolytic, pectinolytic, tannolytic activity.
Bacteria first degraded soil organic materials, and
thenactinobacteria played an important role in degradation of
or-ganic compounds (like cellulose) (USDA 1999). Various
cel-lulolytic and xylanolytic bacteria like Terribacillus
halophilusDurck28, Bacillus nealsonii Durck26, Comamonas
kerstersiiDurck29, Lysinibacillus fusiformis Durck2 and Bacillus
flexusDurck5 known for the aggregations of organic-rich
mattertended to heat up as the indigenous microbial community,
thatrapidly decomposed the utilizable substrates. Kocuria
sp.Durck22 a thermophilic actinobacteria showed a
maximumamylolytic, xylanalolytic and cellulolytic activity at 40
°C.Actinobacteria tended to grow in later stages of composting
Table 5 Effect of compost on tomato shoot length and leaf
size
Treatment Amount (g)
25 50 75 100
Shoot length (cm)
Compost 20.3±0.45 23.6±0.70 25.7±1.20 26.4±0.45
SOM 10.9±0.09 14.5±0.75 19.6±0.63 23.8±0.88
Leaf size (cm)
Compost 10.4±0.65 12.6±0.43 15.3±0.63 14.5±0.85
SOM 7.8±0.70 9.3±0.35 12.1±0.60 10.9±0.99
Data is the mean ± standard error (SE), based on three
independentexperiments with three replicates in each experiment
Table 4 Antagonism in vitro studies
Indicator organism with accession number Test organism with
accession name Diameter (mm)
Bacillus pumilus Durck8 AM778187 Staphylococcus sciuri Durck1
AM778178 5.0
Bacillus subtilis Durck17 AM884573 Bacillus pumilus Durck14
AM778191 1.0
Bacillus subtilis Durck4 AM778181 Bacillus pumilus Durck14
AM778191 and Kocuria sp. Durck22 AM884578 1.0 and 2.0
Bacillus sp. RC2 Staphylococcus sciuri Durck1 AM778178 and
Kocuria sp. Durck22 AM884578 4.5 and 2.5
Bacillus sp. RC1 Bacillus pumilus Durck13 AM778190;
Staphylococcus sciuri Durck1 AM778178and Kocuria sp. Durck22
AM884578
3.0, 6.0 and 7.0
578 Ann Microbiol (2014) 64:571–580
-
and attacked polymers such as hemicellulose, lignin and
cellu-lose (De Bertoldi et al. 1983). The majority of
thermophilicbacteria at 40 °C required several enzymes to decompose
eitherthe simple or the complex compounds tested by
quantitativedetermination and their enzymatic activity were as
xylanolyticfollowed by cellulolytic and then amylolytic,
pectinolytic, pro-teolytic. As the temperature progressed at 50 °C,
high temper-ature favored the cellulose degradation by cellulolytic
bacteria(Bacillus subtilis Durck17), whereas few bacterial isolates
pro-duced proteolytic and pectolytic activity. Stenbro-Olsen
(1998)found that high temperature favored cellulose degradation
bycellulolytic microbes that appear dominant at the end of
thethermophilic stage.
During the cooling and maturation phase, mesophilicbacteria
break down most of the easily degradable materials,and microbes
moving in from the cooler edges recolonizedthe substrate (Hoitink
et al. 1997). The microbial successionof diverse bacterial
communities at cooling and maturationphase degraded xylan,
cellulose and protease (Table 3).However, Atkinson et al. (1996)
studied that most of thebacterial populations in the maturing phase
have proteolytic,amylolytic and cellulolytic capacities.
The genus Bacillus was the most important antagonisticbacterial
group (Ranjbariyan et al. 2011) out of it, Bacillussubtilis was the
most important species, which produced awide range of structurally
related antimicrobial compounds(Arrebola et al. 2010). The extent
of the antibiosis of the twoBacillus isolates against the test
organisms, evaluated in termsof reduced radial growth, was not
similar. The isolatesexhibited a strong activity (inhibition zone
>7 to 1.0 mm)against Gram-positive bacteria as illustrated in
(Table 4). Thestrong activity expressed by a large zone of
inhibition on agarplates indicated, as investigated by Barakate et
al. (2002), thatthose two isolates produce water soluble
antimicrobial metab-olites which may play an important role in the
bio-control ofplant diseases. Sihem et al. (2011) found that the
results ofantibiotics activity expressed in terms of the diameter
of theinhibition zone showed the differences in the percentage
ofantibiosis and specificity of efficacy. Moreover, it may
implythat the investigated Bacillus isolates belonged to
differentspecies or to the same one but they produced different
bioac-tive compounds exhibiting inhibitory activity against a
largenumber of microorganisms.
The development of morphology like shoots length, leafsizes,
fruiting and maturation in tomato plants was favoredafter the
application of nutrient-rich and biologically-activesubstrates like
compost (Table 5). Such changes in the phys-ical properties of the
plant might be responsible for betterplant growth with lower doses
of compost as compared toSOM-based manure.
The difference in photochemical yield of PS II betweenthe
compost and SOM-amended pots at different concentra-tion at
different interval of time. The ratio of Fv/Fm provided
an estimate of the maximum quantum efficiency of PS
IIphotochemistry (Butler 1978). The higher Fv/Fm were ob-served in
75-g compost-amended potted plants at 45 days,which might be
attributed to higher light absorption that wasdue to higher
photochemical efficiency of PS II (Krause et al.1989), whereas the
lower photochemical yield observed inthe SOM amended pots was also
due to the growth irradiance(Fig. 2a).
Various researchers found that ETR was related to themaximum
photosynthetic capacity, reached when the rateof photosynthesis is
limited by the activity of the electrontransport chain or Calvin
cycle enzymes (Ralph et al. 2005).The maximal value was obtained
after 30 days of incubationin the pot amended with 75 g compost.
The increases of ETRwith irradiance were explained by the high
light inducedactivation of the carbon metabolism, which represents
aphotoprotective response against potential photoinhibitioncaused
by excessive light (Ralph et al. 1999). Moreover, byincreasing the
concentration of about 100 g in a different pot,the value was still
lower than in the 75-g amended pot. Thereactive oxygen species
(ROS) induced the inactivation ofthe repair of the photo-damaged
PSII by suppressing the denovo synthesis of the D1 protein
(Nishiyama et al. 2011),which therefore limits the photosynthetic
ETR (Ley andButler 1976). The ETR values revealed another
distinctheterogeneity between compost and SOM amended pots oftomato
plants, whereas the100-g SOM pots had a maximumvalue in just 45
days and then steadily started decreasing(Fig. 2b).
The latter study examined other parameters of
chlorophyllfluorescence as effective photochemical quantum yields
Y(II). Y (II) was directly related to the rate at which
CO2assimilated by the leaf (Genty et al. 1989). The
significantincreased of Y (II) was detected in compost and SOM
duringthe first 15 days and then declined thereafter (Fig. 2c).
Thedeclination may be attributed to the inhibition in
photosyn-thetic CO2 assimilation (Li et al. 2008). The results of
thisstudy confirmed that Y (II) activity was drastically affectedby
compost supplemented pots, while in SOM-amended potsit was largely
deteriorated.
Acknowledgment The authors wish to express their gratitude to
Ms.Urvashi Kuhad, Department of Modern Indian Languages and
LiteraryStudies, University of Delhi, Delhi for editing the
manuscript.
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580 Ann Microbiol (2014) 64:571–580
Molecular...AbstractIntroductionMaterials and methodsIsolation
and enumeration of bacteria during compostingPhylogenetic
analysisEnumeration and determination of culturable bacterial
hydrolase enzyme activity by substrate-specific plate counting
techniquesIn vitro screeningPlant materialEffect of compost on
shoot length, leaf size, fruiting and maturationPhotosynthetic
parametersStatistical analysis
ResultsViable bacteria count and qualitative determination of
bacterial hydrolytic enzyme activity of compost at different
temperaturesMolecular identification of the bacterial isolatesIn
vitro screening of antagonistic activityEffect of compost and SOM
on shoot length, leaf size and fruiting maturation on tomato
plantComplementary changes in Fv/Fm, ETR and Y (II) on tomato
plants leaves
DiscussionReferences