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Isolation and identification of Trichoderma species fromdifferent habitats and their use for bioconversion of solid waste
Ahsanur RAHMAN1, Most. Ferdousi BEGUM1, Matiur RAHMAN1, M. A. BARI1,G. N. M. ILIAS2, M. Firoz ALAM1
1Department of Botany, University of Rajshahi, Rajshahi 6205, BANGLADESH2Timber Products and Environment Monitoring Cell, Rural Electrification Board (REB),
Shiromoni, Khulna 9204, BANGLADESH
Received: 20.05.2009
Abstract: The native population of Trichoderma species collected from different habitats was quantified and characterised.The identified strains are T. harzianum (IMI-392432, 392433, 392434); T. pseudokoningii (IMI-392431) and T. virens(IMI-392430). Out of five strains, T. harzianum was the most common in all of the habitats. Colony forming units (cfu’s)of Trichoderma species varied significantly (P = 0.05) in different habitats and were positively correlated with the physico-chemical characteristics of the habitat. Trichoderma was found to have adapted to an acidic habitat. In testing for anefficient bioconversion agent, T. harzianum (IMI-392432) was found to be the most effective in kitchen wastedecomposition. It provided the highest volume (31.80%) and weight (30.80%) losses in waste treated with sporesuspension. Promising results were also noted using a combined treatment with different strains/species of Trichoderma,which resulted in 18% greater decomposition of waste than the control.
Farklı habitatlardan Trichoderma türlerinin izolasyonu ve tanımlanması ve katıatıkların biyolojik dönüşümü için kullanımı
Özet: Farklı habitatlardan toplanan Trichoderma spp.’nin yerli populasyonlar karakterize edilmiş ve miktarlarıbelirlenmiştir. Tanımlanan suşlar, T. harzianum (IMI-392432, 392433, 392434); T. pseudokoningii (IMI-392431) ve T.virens (IMI-392430) olarak isimlendirilmiştir. Beş suş arasından, T. harzianum bütün habitatlarda en yaygın olanıdır.Trichoderma türlerinin koloni oluşturan birimleri (cfu’ler) farklı habitatlarda önemli bir şekilde (P = 0,05) değişmiş veolumlu bir şekilde habitatın fiziko-kimyasal özellikleri ile ilişkilendirilmiştir. Trichoderma’nın asidik bir habitata adapteolduğu bulunmuştur. Etkili biyolojik dönüşüm ajanı denenerek mutfak atığı ayrıştırmasında en etkili türün T. harizanumolduğu bulunmuştur. Spor süspansiyonu ile muamele edilen atıklardaki en yüksek hacim (% 31,80) ve ağırlık kayıpları(% 30,80) bulunmuştur. Gelecek vaadeden sonuçlar, kontrolden %18 daha fazla ayrıştırma ile sonuçlanan Trichoderma’nınfarklı suş ve türleri ile kombine edilmiş bir uygulama kullanılarak da bildirilmiştir.
IntroductionTrichoderma species are cosmopolitan fungi,
frequently present in all types of soil, manure anddecaying plant tissues (1). Their dominance in soilmay be attributed to their diverse metabolic capabilityand aggressive competitive nature (2). Thesecharacteristics make them significant decomposers ofwoody and herbaceous material. Trichoderma speciesare also able to degrade domestic waste relativelyquickly without emitting bad odours (3). Among thevarious global environmental hazards, municipalsolid waste (MSW) is one of the greatest modernproblems. Every day large quantities of domestic,kitchen, and municipal waste are generated indeveloped and developing countries (4). Propermanagement and safe disposal of MSW is a major,complex problem that can affect the air, land, andwater. Therefore, attention has been focused on non-hazardous, environmentally friendly, sustainabletechniques involving bioconversion or biologicallybased treatment of domestic waste to overcome thisserious problem (5). Microorganisms from relevantenvironments possess greater degradation capabilitieson related wastes in the biodegradation process (6).Thus, bioconversion or biodegradation might be apotentially effective measure for proper managementof municipal solid waste. Therefore, the presentinvestigation was undertaken to isolate, identify, andquantify Trichoderma species from different habitatsof the Rajshahi metropolitan area and the premises ofthe compost plant of the Natore Development Society(NDS). The physico-chemical characteristics of thesamples were analysed and correlations betweensample characteristics and colony forming units (cfus)were determined. The biodegradative potential ofTrichoderma isolates was also evaluated on kitchensolid waste in vitro.
Materials and methodsSample collection Different types of samples such as soil, humus,
kitchen waste, and compost were collected fromdifferent sites in the Rajshahi metropolitan and itssuburban areas, and on the premises of the NDScompost plant in Natore from April to July 2003. Soilsamples were collected from the different sites (e.g.,
Botanical garden, the 3rd Science building and theAgriculture Research Field of Rajshahi University(RU), Budpara, and agriculture field at Khorkhori,Rajshahi Metropolitan City). Humus was collectedfrom the Rajshahi City Corporation dumping site,Kashiadanga. Kitchen wastes were collected fromdustbins in Rokeya Hall, Nawab Abdul Latif Hall, theTeachers Quarter, the Railway Station, BangabandhuHall and the IBS quarters of RU Campus, and fromthe Binodpur Bazaar, the Kazla Bazaar, the ShahebBazaar and the Science lab quarters of Rajshahi CityCorporation. Compost was collected from thecompost plant and compost heap of NDS, Natore. Thesamples were taken from a 15 cm depth and collectedin sterile polyethylene bags, which were transportedto the laboratory and stored at 4 °C until use.
Isolation, identification, and quantification ofTrichoderma spp.
For isolation of Trichoderma strains, a serialdilution technique was followed and a 103 dilution ofeach sample was prepared. One millilitre of eachsolution was pipetted onto a Rose Bengal Agar (RBA)plate and incubated at 28 °C for 1 week. The cultureplates were examined daily and each colony thatappeared was considered to be one colony formingunit (cfu). After enumeration of cfu, individualcolonies were isolated from the same plates and eachuncommon colony was reisolated onto a fresh PotatoDextrose Agar (PDA) plate. Distinct morphologicalcharacteristics were observed for identification, andthe plates were stored at 4 °C.
Two techniques, visual observation on petri dishesand micro-morphological studies in slide culture,were adopted for identification of Trichodermaspecies. For visual observation, the isolates weregrown on PDA agar for 3-5 days. The mode of myceliagrowth, colour, odour and changes of medium colourfor each isolate were examined every day. For micro-morphological studies, a slide culture technique wasused (6). Examination of the shape, size, arrangementand development of conidiophores or phialidesprovided a tentative identification of Trichoderma spp.Samples were compared to a taxonomic key for thegenus Trichoderma (7) and further verified andconfirmed by CABI, Bioscience, Surrey, UnitedKingdom.
Isolation and identification of Trichoderma species from different habitats and their use for bioconversion of solid waste
184
Estimation of moisture content and acidity (pH)of the samples
Freshly collected samples were placed in glasstubes and the initial weights were recorded. Thesamples were then placed inside a hot-air incubatorat 110 °C until constant weights were achieved. Themoisture content was calculated using the followingformula provided by American Wood PreservationAssociation’s Standards (AWPA) (8):
MC (%) = W – w / W × 100 where MC is moisture content, W is the initial weightand w is the constant weight after oven drying.
The acidity of the samples (pH) was measuredusing the method of Black (9).
Chemical analysis of the samplesThe chemical characteristics of all the samples
were analysed by the Soil Analysis Laboratory, SoilResource Development Institute, Regional Office,Shampur, Rajshahi, for the following: organic matter(%), total nitrogen (N) (%), phosphorus (P) (mg/g),and potassium (K) (mL/100 g). Nitrogen wasdetermined by the micro Kjeldhal method. AvailableP was determined using the Modified Olsen method,K was determined using the N NH4OAC method, andorganic matter was determined using the drycombustion method (10).
Preparation of organic solid waste forbioconversion
Kitchen wastes were collected from differentgarbage centres on the RU Campus in polythene bags.Samples were cut into 2-3 mm pieces and 50 g of eachwas aliquotted into a 150 mL bottle, which was thensealed with a cotton plug. The bottles containingkitchen solid waste were then autoclaved at 121 °Cand 1.05 kg/cm2 pressure for 15 min.
Preparation and application of culture discsTrichoderma strains were cultured on potato
dextrose agar (PDA) medium at 30 °C for 48 h. Thena culture disc (5 mm diam.) was cut with a cork borerand 6 culture discs were inoculated with autoclavedkitchen waste. Control treatments were performedwith no inoculation. A sterile thermometer was alsopushed through the cotton plug of each bottle. Thebottles were then sealed with Para film and labelled.
The entire process was completed inside a laminarflow hood.
Preparation and application of sporesuspensions of Trichoderma
Ten millilitres of autoclaved distilled water wasadded to a 4-day-old Trichoderma culture tube andmixed well. Next, 5 mL of this spore suspension wasadded to each autoclaved kitchen waste bottle andmixed well. For the consortium, 1 mL of sporesuspension of each strain (total 5 mL) was used.Control treatments were also performed with noinoculation. As with the culture disc experiment,sterile thermometers were used and the bottles weresealed with Parafilm.
Changes in odour, temperature, volume loss andweight loss of the decomposed organic solid wastewere observed at 10-day intervals up to 30 days. Formeasurement of volume loss (%), the followingformula was used:
Volume loss (%) = V-V1/V × 100 where V is initial volume, V1 is final volume.
Weight loss (%) of decomposed garbage wascalculated employing the following formula:
Weight loss (%) = W-W1/W × 100where W is initial weight, W1 is final weight.
Statistical analysisThe experiment was conducted using a complete
randomised design (CRD) with 3 replications.Correlations were calculated between the sample’sphysico-chemical characteristics and cfus. The datawere statistically analysed with the help of thecomputer package MSTAT-C and also tested byDMRT (11).
Results and discussion Isolation and identification of Trichoderma spp.In this study, a total of 135 isolates of Trichoderma
were isolated from different samples (Table 1). On thebasis of morphological characteristics, these 135isolates of Trichoderma were divided into 5 aggregategroups (Table 2). Representative isolates from eachgroup were sent for identification to CABIBiosciences, Surrey, United Kingdom, and the
A. RAHMAN, M. F. BEGUM, M. RAHMAN, M. A. BARI, G. N. M. ILIAS, M. F. ALAM
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Isolation and identification of Trichoderma species from different habitats and their use for bioconversion of solid waste
186
Table 1. Frequency of isolation of Trichoderma species and their sources of origin.
Sources of Trichoderma spp. Sample types Species name and isolate number No. of isolates
T. harzianum (SBT-190, SBT-191, SBT-192. SBT-193, SBT-194). 5BHRU(Bangabandhu Hall, RU). Kitchen waste T. harzianum (RUT-195, RUT-196, RUT-197). 3MHRU (Mannuzan Hall, RU). Kitchen waste T. harzianum (RUT-104, RUT-199, RUT-200, RUT-201). 4
T. pseudokoningii. (RUT-202, RUT-203, RUT-204, RUT-205, RUT-206, RUT-207). 6
ARF (Agriculture research field, RU). Soil T. harzianum (RUT-208, RUT-209, RUT-210). 3IBSQRU (IBS quarter, RU). Kitchen waste T. harzianum (RUT-211, RUT-212, RUT-213, RUT-214). 4BPR, (Budpara, Rajshahi). Soil T. harzianum (BPRT-215, BPRT -216, BPRT -217, BPRT -218). 4TQPRU (Teacher quarter pubapara, RU). Kitchen waste T. harzianum (RUT-219, RUT-220, RUT-221). 3CCDS (City corporation dumping site, Humus T. virens (CCDKT-222, CCDKT -223, CCDKT-224, CCDKT-225). 4Kashiadanga, Rajshahi). T. pseudokoningii (CCDKT-226, CCDKT-227). 2
T. harzianum (CCDKT-228, CCDKT-229, CCDKT-230). 3KR (Agriculture field Khorkhori, Rajshahi.). Soil T. harzianum (AKRT-231, AKRT-232). 2SLQ (Science Lab Quarter, Rajshahi). Kitchen waste T. harzianum (SLQT-233, SLQT-234, SLQT-235). 3NDS (Natore Development Society, Natore) Compost T. harzianum (NDST-236, NDST-237, NDST-238, NDST-239). 4NDS (Natore Development Society, Natore). Compost T. harzianum (NDST-105, NDST-240, NDST-241, NDST-242, NDST-243). 5
Total isolates = 135
number of Trichoderma groups was reduced to 3species. These species of Trichoderma were identifiedas T. harzianum Rifai (IMI -392432, 392433& 392434)(86 isolates), T. pseudokoningii Rifai (IMI-392431) (27isolates), and T. virens (J.H. Mill. Gidden & A.A.Foster) Arx. (IMI-392430) (22 isolates) (Figure 1).The identified Trichoderma species are characterised
in Table 2. T. harzianum was the most frequentlyisolated species, and it was recovered in nearly all thesamples. In a similar study, Zakaria (12) isolated andidentified 5 species of Trichoderma from therhizosphere of rubber trees in Malaysia, namely T.hamatum (Bon). T. harzianum Rifai, T. koningii(Oudemans), T. longibrachiatum Rifai, and T. viride
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Table 2. Characterisation and identification of Trichoderma species isolated from different habitats.
Group and Trichoderma spp. Sources ofStrain with samples Macro-/Microscopic characteristics
IMI No.
Initially the colony colour was observed to be whitish to light green, watery in centre. Next the colony
Group-1 gradually became deep grass green in colour and looked soft T. virens (IMI -392430) Humus, and leathery to the naked eye. The conidiophores were erect,
RUT-101 BG, RU smooth, penicillately branched; asymmetrical branches singly or vertically arranged at different levels, phialides were flask-shaped, coverage toward the main branch, emphasizingthe penicillate branching. Phialospores were sub-globose to elliptical, smooth-walled. (Figure 1, 1a & 1b).
Whitish to pale green, hairy and flappy mycelial mat uniformly Group-2 grown in PDA plate at 3-4 days. Next pale green turned into whitish
T. pseudokoningii (IMI -392431) Kitchen waste, green to dark green colour. Branched conidiophores and dendroid RUT-102 RH, RU conidiophores terminated by phialides carried confused ellipsoids to
subglobose phialospores. Phialospores were pigmented, smooth, elliposide up to a little more than 4 mm long (Figure 1, 2a&2b).
At the early stage whitish to greenish mycelia appeared. Group-3 Next a deep green colour developed in central part and gradually
T. harzianum (IMI -392432) Soil, extended to the periphery. Finally, it appeared a whitish green colour. RUT-103 3SB, RU Mostly globose conidia developed on phialides produced in the
opposite direction in each point. (Figure 1, 3a & 3b).
The colony colour was initially watery white and turned bright green Group-4 to dark green and dull green with compact conidiophores
T. harzianum (IMI -392433) Compost, throughout the petriplates. Reproductive structures were closely RUT-104 MH, RU similar to RUT-103. (Figure 1, 4a& 4b).
The colony was observed to be watery white at the early stage (3-4 days),Group-5 (IMI-392434) Compost heap, bright green and dark green coloured mycelia mat exhibited at the
T. harzianum NDS, Natore central part of PDA plate at the later stage. Reproductive structuresNDST-105 were very similar to RUT-103. (Figure 1, 5a&5b).
Isolation and identification of Trichoderma species from different habitats and their use for bioconversion of solid waste
188
Figure 1. Photograph shows colonies of Trichoderma strains on PDA plate (dorsal view) andconidiophore with conidia. 1a & 1b. T. virens (IMI-392430), 2a & 2b. T. pseudokoningii(IMI-392431), 3a & 3b. T. harzianum (IMI-392432), 4a &4b. T. harzianum (IMI-392433) and 5a & 5b.T. harzianum (IMI-392434).
Per.ex S.F. Gray. Ilias (13) isolated 4 species ofTrichoderma in healthy and diseased oil palmrhizosphere in Malaysia, namely T. harzianum, T.aureoviride, T. longibrachiatum, and T. virens. Thesewere also identified following the classification ofRifai (7) but verified by IMI, England. It was exhibitedthat T. harzianum was the most abundant species.Okoth et al. (14) isolated 306 Trichoderma strainsfrom 60 soil samples from different places andobserved T. harzianum to be the most frequentlyisolated species, but in coffee farms, T. viride was themost frequently identified species. T. citrinoviride wasthe most common in Napier farms. The present resultis accordance with the above findings.
Quantification and distribution of TrichodermaTrichoderma spp. were recovered from different
habitats. The quantity of the fungi obtained was high,but unequal numbers of the various species werefound in different samples. Colony forming units(cfus) of Trichoderma isolates from the differenthabitats were significantly (P = 0.05) different (Table3 and Figure 2). Populations of Trichoderma speciesranged from 13.31 ± 0.28 to 34.68 ± 2.86 × 103/g inkitchen waste, 17.17 ± 0.39 to 35.32 ± 2.27 × 103/g insoil, 16.68 ± 1.46 × 103/g in humus, and 35.48 ± 2.88× 103 to 36.67 ± 3.66 × 103/g in compost and compostheap. The highest number of cfus (36.67 ± 3.66 ×103/g) was found in compost (NDS) and the lowestnumber of cfus (13.23 ± 0.87 × 103/g) was recorded inthe kitchen waste of Bangabandhu Hall, RajshahiUniversity. The identified Trichoderma species wererandomly distributed in different habitats.
Species of Trichoderma are distributed worldwide.They are generally found in soils, including foresthumus layers and agricultural and orchard soils (15).At the species level, however, individual speciesaggregates may be restricted in their geographicdistribution. Trichoderma populations weresignificantly different for the various sites on RajshahiUniversity campus and in its suburban area. Therewas a significant difference in distribution ofTrichoderma species among the different habitats.Seasonal variation is a factor that affects the numberof Trichoderma species in the samples. Greaterpopulations of Trichoderma species in soils have beenestimated in the spring and summer months ascompared to the autumn and winter (16). Danielson
and Devey (17) found that T. viride and T. polysporumare restricted to areas where low temperatures prevail.On the other hand, T. harzianum is most commonlyfound in warm climate regions. T. hamatum and T.koningii are widely distributed in areas of diverseclimatic conditions (15). In the present study, thesamples were collected in summer (April to July),which is in partial support of the above findings.
Effects of the physical and chemicalcharacteristics of the samples on the occurrence ofTrichoderma species
Soil moisture greatly affected the population (cfus)of Trichoderma species. In the present study, themoisture content of the samples was high, and apositive and significant (P = 0.05) correlation (datanot shown) was observed between the moisturecontent and cfus of almost of the samples. Highmoisture content might be helpful to increase thepopulation of Trichoderma. Populations ofTrichoderma spp. have been reported to be greater inmoist soils or even in soils with excessive moisture ascompared to dry soils (17). Population densities ofTrichoderma were found to be positively associatedwith sample moisture when soil samples werecollected throughout the year in a California alfalfafield (18). Therefore, it may be noted that the densityof the Trichoderma population is dependent on themoisture of habitats.
The results revealed that the pH level in differenthabitats was moderately acidic, and there was apositive correlation between acidity and thepopulation (cfus) of Trichoderma, although this wasnon-significant (P = 0.05). Soil acidity has previouslybeen reported as a factor affecting the presence anddensity of Trichoderma spp. (19). T. viride and T.polysporum Rifai were more prevalent in acidicconiferous forest soils, whereas T. hamatumpredominated in more neutral deciduous forest soils(20), and T. viride predominated in acidic pine forestsoil (21). Therefore, it may be considered that somespecies of Trichoderma may be adapted to acidichabitats.
The sample’s key nutrients, organic matter,nitrogen, phosphorus, and potassium, displayed somevariations with respect to the distribution andabundance of Trichoderma (14). In this study, organic
A. RAHMAN, M. F. BEGUM, M. RAHMAN, M. A. BARI, G. N. M. ILIAS, M. F. ALAM
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Isolation and identification of Trichoderma species from different habitats and their use for bioconversion of solid waste
190
Tabl
e 3. L
ocat
ion,
sour
ces,
moi
sture
cont
ent,
pH, a
nd ch
emic
al ch
arac
teris
tics o
f diff
eren
t sam
ples
.
Sam
ple n
o.Sa
mpl
e So
urce
s of
Moi
sture
pH
Org
anic
To
tal N
(%)
PK
No.
of c
fu’s
loca
tion
sam
ple
cont
ent (
%)
mat
ter (
%)
μg/g
Ml/1
00 g
(103 /g
of s
ampl
es)
S/1
BG, R
USo
il46
.58
± 5
.44
ab6.
43 ±
0.2
6 bc
de8.
6 ±
0.1
j0.
23 ±
0.0
2 e
55.8
4 ±
5.3
2 k
0.51
± 0
.03
j20
.67
± 0
.45
efg
S/2
RH, R
UKi
tche
n W
aste
44.0
7 ±
5.61
ab6.
35 ±
0.2
2 de
13.4
± 0
.06
ef0.
78 ±
0.0
3 c
214.
67 ±
1.5
1 f
1.58
± 0
.75
f24
.59
± 0.
33 cd
e
S/3
3sb,
RU
Soil
47.5
5 ±
3.48
ab6.
2 ±
0.38
efg
8.22
± 0
.92
j0.
32 ±
0.0
5 de
79.2
3 ±
1.94
ij0.
54 ±
0.0
4 i
17.1
7 ±
0.39
fgh
S/4
NA
LH, R
UKi
tche
n w
aste
44.6
2 ±
0.36
ab5.
9 ±
0.15
fgh
12.2
± 0
.26
fg0.
49 ±
0.0
4 cd
e15
0.5
± 5.
43 g
0.56
± 0
.08
h22
.67
± 0.
97 d
ef
S/5
TQW
P, RU
Kitc
hen
was
te48
.58
± 0.
48 ab
5.7
± 0.
13 h
ij15
.3 ±
0.1
de
0.79
± 0
.02
c13
9.74
± 4
.93
g1.
58 ±
0.1
4 f
19.3
7 ±
0.98
efg
S/6
RSRU
Kitc
hen
was
te41
.47
± 0.
38 ab
6.86
± 0
.04
ab16
.51
± 2.
01 d
1.2
± 0.
06 b
214.
33 ±
6.4
3 f
1.04
± 0
.33
g26
.68
± 1.
28 cd
S/7
BB, R
UKi
tche
n w
aste
43.2
2 ±
4.92
ab5.
76 ±
0.0
8 gh
i11
.61
± 0.
08 fg
h0.
37 ±
0.0
2 de
35.8
6 ±
7.09
l1.
41 ±
0.0
3 f
13.3
1 ±
0.28
h
S/8
KRKi
tche
n w
aste
47.0
8 ±
1.29
ab6.
77 ±
0.0
3 ab
c18
.91
± 0.
13 b
c1.
63 ±
0.3
7 a
261.
42 ±
1.2
6 e
3.96
± 0
.21
ef30
.2 ±
1.2
2 bc
S/9
SBKi
tche
n w
aste
36.9
9 ±
1.26
bcd
5.3
± 0.
05 k
8.91
± 0
.22
ij0.
27 ±
0.0
5 e
60.9
3 ±
1.16
jk0.
46 ±
0.0
2 k
16.3
1 ±
1.06
gh
S/10
BHRU
Kitc
hen
was
te37
.64
± 3.
15 ab
cd5.
33 ±
0.0
8 jk
8.14
± 0
.52
j0.
22 ±
0.1
5 e
62.6
7 ±
2.64
ijk
0.39
± 0
.03
l13
.23
± 0.
87 h
S/11
MH
RUKi
tche
n w
aste
45.1
9 ±
4.26
ab6.
67 ±
0.0
3 ab
cd19
.62
± 0.
28 ab
1.6
± 0.
06 a
1138
± 4
.12
abc
4.04
± 0
.62
ef34
.33
± 2.
87 ab
S/12
ARF
Soil
41.4
7 ±
2.81
ab6.
9 ±
0.12
a21
.82
± 0.
24 a
1.72
± 0
.01
a11
45 ±
9.8
4 ab
c24
.56
± 0.
08 b
35.3
2 ±
2.27
ab
S/13
IBSQ
RUKi
tche
n w
aste
43.5
3 ±
4.62
ab5.
8 ±
0.06
ghi
12.6
1 ±
0.05
fg0.
61 ±
0.0
2 cd
109.
9 ±
6.26
h7.
35 ±
1.5
8 de
24.3
1 ±
2.16
de
S/14
BPR
Soil
48.7
7 ±
2.58
a6.
7 ±
0.12
abcd
17.2
4 ±
0.58
cd1.
21 ±
0.0
6 b
1128
.33
± 4.
09 b
c21
.21
± 3.
46 b
c34
.46
± 2.
62 ab
S/15
TQPU
RKi
tche
n w
aste
49.3
6 ±
4.12
a5.
7 ±
0.11
hijk
10.5
1 ±
0.03
ghi
0.23
± 0
.58
e21
9.76
± 4
.67
f17
.43
± 3.
56 c
27.2
8 ±
3.56
cd
S/16
CCD
SH
umus
28.7
6 ±
5.36
d5.
4 ±
0.12
ijk
9.84
± 0
.15
hij
0.26
± 0
.02
e83
.17
± 3.
18 i
10.9
6 ±
2.29
d16
.68
± 1.
46 g
h
S/17
KRSo
il30
.29
± 3.
19 cd
5.9
± 0.
1 fg
h16
.58
± 0.
49 d
1.32
± 0
.02
b11
00 ±
1.6
3 d
20.7
9 ±
3.23
bc
33.3
2 ±
2.94
ab
S/18
SLQ
Kitc
hen
was
te39
.59
± 4.
88 ab
c6.
5 ±
0.05
abcd
16.6
5 ±
1.02
cd1.
58 ±
0.8
9 a
1127
.33
± 4.
08 c
23.2
7 ±
3.62
b34
.68
± 2.
86 ab
S/19
ND
SC
ompo
st47
.58
± 4.
83 ab
5.9
± 0.
12 fg
h19
.85
± 0.
82 ab
1.72
± 0
.19
a11
58.3
3 ±
0.88
a31
.02
± 2.
93 a
36.6
7 ±
3.66
a(C
ompo
st pl
ant)
S/20
ND
SC
ompo
st45
.34
± 3.
26 ab
6.3
± 0.
27 d
ef19
.96
± 0.
89 ab
1.68
± 0
.04
a11
49.6
7 ±
5.21
bb
35.8
4 ±
2.34
a35
.48
± 2.
88 ab
(Com
post
heap
)
Sam
e let
ters
with
in a
colu
mn
are n
ot si
gnifi
cant
ly d
iffer
ent b
y D
MRT
at th
e 5%
leve
l.BG
=Bot
anic
al g
arde
n, R
U; R
H=R
okey
a H
all,
RU; 3
SB=
3rdsc
ienc
e bu
ildin
g RU
; NA
LH=
Naw
ab A
bdul
Lat
if H
all,
RU; T
QW
P= T
each
ers q
uart
er w
est p
ara,
RU; R
SRU
= Ra
ilway
stat
ion,
RU
; BB=
Bino
dpur
Baz
aar,
Rajsh
ahi;
KR=
Kazla
, Raj
shah
i; SB
= Sh
aheb
Baz
ar, R
ajsh
ahi;
BHRU
= Ba
ngab
andh
u H
all,
RU; M
HRU
= M
annu
zan
Hal
l, RU
; ARF
= A
gric
ultu
re re
sear
ch fi
eld,
RU
; IBS
Q=
IBS
quar
-te
r, RU
; BPR
= Bu
dpar
a, Ra
jshah
i; TQ
PRU
= Te
ache
rs q
uarte
r pur
bapa
ra, R
U; C
CDS=
City
corp
orat
ion
dum
ping
site
, Rajs
hahi
; KR=
Kho
rkho
ri, R
ajsha
hi,S
LQ=S
cien
ce L
ab q
uarte
r, Ra
jshah
i; N
DS=
Na-
tore
Dev
elop
men
t Soc
iety
,Nat
ore.
matter showed a significant (P = 0.05) and positivecorrelation with the population of Trichoderma spp.(data not shown). Nitrogen showed a non-significant(P = 0.05) positive correlation with the occurrence ofTrichoderma species in almost of all the habitats (datanot shown). Trichoderma spp. have been recoveredmore frequently from different sources with highorganic matter content (22). Nitrate-nitrogen is not apreferred nitrogen source for this genus (16). Thepresent result is completely in accordance with thefindings of these studies. The results of the currentstudy further show that P and K had a positive andsignificant (P = 0.05) correlation with Trichodermaspecies (data not shown). Okoth et al. (14) observedthe same results with the most common species ofTrichoderma, i.e. T. harzianum and T. viride.
Bioconversion of solid wasteFungi play an important role in the decomposition
of organic waste and can be important contributorsto optimal agricultural waste bioconversion (23).Among the 5 strains studied, T. harzianum (IMI-392432) showed the highest volume (31.81%) andweight (31.80%) losses using spore suspension (Table4). Using culture discs, the highest volume (22.72%)and weight (28.03%) losses were also observed in T.harzianum (IMI-392432). Zheng and Shetty (24)reported that Trichoderma is capable of producingvarious polysaccharide degrading enzymes, whichmay help to degrade long chain carbon compounds,particularly the bulking materials that normallycomprise sewage sludge. Martin and Dale (25) usedweight loss measurement to estimate the tissuedegradation of Bermuda grass (Cynodon dactylon L.)
stolons and Zoysia grass (Zoysia japonica Steud,‘Mayer’) stolons. Bari et al. (26) reported 31.80% and28.03% weight loss when culture pellets and sporesuspensions of T. harzianum were used, respectively.
Combined treatment with the Trichoderma strainshad promising effects in the decomposition of organicsolid waste, resulting in a greater loss (18%) of theoriginal material than the control. Bari et al. (26)reported that combined treatment was the mosteffective method, promoting 23% greater weight lossthan the control in their experiments. Thus, thecomposition of the microbial culture affects thequality of the compost. In another previous study,mixed microbial inoculants comprised of Trichurusspirals, Aspergillus, and Paccilomyces fusisporusaccelerated the process of decomposition, resulting ina 10% greater loss than the control in 12 weeks ofdecomposition (27).
ConclusionFrom the above findings, it can be concluded that
a significant number of Trichoderma species occupydifferent habitats. Some species of Trichoderma werefound to be adapted to acidic habitats. Thepopulations (cfus) are significantly and positivelycorrelated with moisture, organic matter, phosphorus,and potassium, but non-significantly and positivelycorrelated with pH and nitrogen in almost all of thesamples. The most frequently isolated species was T.harzianum, and the strain T. harzianuum IMI- 392432was identified as an effective agent for solid wastebioconversion using spore suspension. The co-culture
A. RAHMAN, M. F. BEGUM, M. RAHMAN, M. A. BARI, G. N. M. ILIAS, M. F. ALAM
191
BG RH3S
BN
ALH
TQW
P
RSRUBB K
RSB
BHRU
MH
RUARF
IBSQ
RUBP
R
TQPRU
CCD
SKR
SLQ
ND
SCO
M
ND
SCH
Location of Trichoderma isolates
Thic
ho
der
ma
po
pu
lati
on
(C
FU
/10
3g)
4540353025201510
50
cfu’s of Trichoderma spp.
Figure 2. Comparison of Trichoderma population with different habitats.
Isolation and identification of Trichoderma species from different habitats and their use for bioconversion of solid waste
192
Tabl
e 4. C
hang
es in
tem
pera
ture
, vol
ume l
oss,
and
wei
ght l
osso
f sol
id w
aste
usin
g di
ffere
nt T
richo
derm
a iso
late
s
Stra
inCh
ange
s of c
hara
cter
istic
s of s
olid
was
te af
ter 1
0 da
ys
Chan
ges o
f cha
ract
erist
ics o
f sol
id w
aste
afte
r 20
days
Chan
ges o
f cha
ract
erist
ics o
f sol
id w
aste
afte
r 30
days
Tem
pera
ture
Vo
lum
e W
eigh
tTe
mpe
ratu
reVo
lum
eW
eigh
tTe
mpe
ratu
reVo
lum
eW
eigh
t(°
C)lo
ss· (
%)
loss
• (%
)(°
C)lo
ss• (
%)
loss
· (%
)(°
C)lo
ss• (
%)
loss
· (%
)
CD29
4.72
±0.
094
10.9
2±0.
296
SPS
3014
.81
±0.1
2517
.68
±0.3
17SP
S29
20.7
2 ±1
.320
24.1
4±0.
529
T.vi
rens
(IM
I-39
2430
)SP
S28
6.95
±0.1
4911
.82±
0.28
4CD
3018
.99
±0.1
5420
.88
±0.4
04CD
2828
.32
± 0.
526
27.9
0±0.
264
CD29
4.45
±0.0
5910
.23±
0.21
8SP
S30
13.6
3 ±0
.107
16.5
4 ±0
.581
SPS
2918
.81±
0.49
123
.28±
0.36
0T.
pse
udok
onin
gii.
(IM
I-39
2431
)SP
S28
6.25
±0.5
5412
.58±
0.57
7CD
3018
.72
± 0.
102
22.1
8 ±0
.417
CD28
2830
±0.
644
28.0
2±0.
721
CD29
5.18
±0.1
3912
.44±
0.88
1SP
S30
17.2
7 ±0
.105
21.0
0 ±0
.577
SPS
2922
.72±
0.41
528
.03±
0.79
3T.
har
zian
um (I
MI-
3924
32)
SPS
287.
63±0
.137
14.9
9±0.
577
CD30
20.4
5 ±0
.112
23.8
6 ±0
.404
CD28
31.8
1±0.
515
31.8
0±0.
529
CD29
4.45
±0.1
0611
.66±
0.15
2SP
S30
15.5
4 ±0
.105
19.2
3 ±0
.138
SPS
2922
.45
±0.2
6425
.98±
0.21
4T.
har
zian
um (I
MI-
3924
33)
SPS
285.
63±0
.126
13.2
5±0.
665
CD30
19.7
2 ±0
.122
20.8
8 ±0
.346
CD28
30.0
8 ±0
.567
28.8
8±0.
105
CD29
6.18
±0.1
1511
.24±
0.33
3SP
S30
14.5
4 ±0
.107
19.9
2 ±0
.208
SPS
2921
.73±
0.39
625
.76±
0.43
5T.
har
zian
um (I
MI-
3924
34)
SPS
286.
90±0
.189
13.4
4±0.
305
CD30
20.7
2 ±0
.112
21.4
4 ±0
.321
CD28
29.3
5±0.
366
29.0
4±0.
142
T.h(
IMI-3
9243
2,39
2433
,392
434)
SPS
288.
90±0
.132
15.8
7±0.
120
SPS
3126
.45
±0.1
1524
.82
±0.5
85SP
S29
33.3
5 ±0
.199
36.5
6±0.
624
+ T.
v (I
MI-
3924
30)+
T. p
(IM
I-39
2431
)C
ontro
l-
273.
45±0
.463
8.02
±0.1
11-
278.
18 ±
0.52
315
.06±
0.75
0-
2717
.36±
0.46
618
.56±
0.32
1
•Mea
n of
3 re
plic
atio
ns, S
PS=
Spor
e sus
pens
ion,
CD
= Cu
lture
disc
.T.
h= T
richo
derm
a ha
rzia
num
, T. v
= T
richo
derm
a vi
rens
, T. p
= T
richo
derm
a ps
eudo
koni
ngii.
A. RAHMAN, M. F. BEGUM, M. RAHMAN, M. A. BARI, G. N. M. ILIAS, M. F. ALAM
193
use of different species and strains of Trichodermaresulted higher and quicker ways of solid wastedecomposition than the use of a single species orstrain.
AcknowledgementThe authors express thanks to the Ministry of
Science, Information and CommunicationTechnology, Bangladesh, for financial support, and
are also grateful to CABI Bioscience, U.K., for theidentification of Trichoderma strains.
Corresponding author:Most. Ferdousi BEGUMDepartment of Botany, University of Rajshahi,Rajshahi 6205, BANGLADESHE-mail: ferdrita @yahoo.com
0
5
10
15
20
25
30
35
40
BG RH
3SB
NA
LH
TQ
WP
RSR
UBB K
RSB
BHRU
MH
RU
ARF
IBSQ B
PR
TQ
PRU
CC
DS
KR
SLQ
ND
SC
OM
ND
SCH
Location of Trichoderma isolates
Tri
ch
od
erm
a p
op
ula
tio
n (
CF
U/1
03 g
)
0
200
400
600
800
1000
1200
1400
Ph
ysi
cal
an
d c
hem
ical
ch
ara
cte
rist
ics
of
the s
am
ple
s
cfu N P K pH om
Figure 3. Effect of physico-chemical characteristics on the occurrence of Trichoderma spp.BG=Botanical garden; RH=Rokeya Hall; 3SB= 3rd science building; NALH= Nawab AbdulLatif Hall; TQWP= Teachers quarter west para; RSRU= Railway station; BB= BinodpurBazaar; KR= Kazla Rajshahi; SB= Shaheb Bazaar; BHRU= Bangabandhu Hall; MHRU=Mannuzan Hall; ARF= Agriculture research field; IBSQ= IBS quarter; BPR= BudparaRajshahi; TQPRU= Teachers quarter purbapara, RU; CCDS= City corporation dumping site;KR= Khorkhori Rajshahi; SLQ= Science Lab quarter; NDSCOM= Natore DevelopmentSociety Compost plant;. NDSCH=Natore Development Society Compost Heap.
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Isolation and identification of Trichoderma species from different habitats and their use for bioconversion of solid waste