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: kuhad85@gmail.com

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

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:

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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

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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).

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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

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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

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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

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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

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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