3rd International Conference on Chemical, Biological and Environment Sciences (ICCEBS'2013) January 8-9, 2013 Kuala Lumpur (Malaysia)
Role of Bacteria and Mold as Agent
Plant Litter Composting
Ardhiani Kurnia Hidayanti, Nungki Amalia Puspa Kirana, Endang Sutariningsih Soetarto
composting. The research was command by collecting and
composting leaf litters from Biological Forest and Biology
Field Station Gadjah Mada University, Indonesia. Selection
isolate that had been isolation from leaf litters compos based
on they capability on degrading of cellulose (CMC and
Avicel), Hemicelluloses (Xylan), and Lignin (Tanin). The
highest cellulose activity on cellulose agar substrate were
selected for further composting experiment.
The study comprise of two phase. First phase, leaf litter
were collected then composting, , strains from compos sample
were isolated onto selective medium. Second phase is
composting experiment, the best selected isolates were
inoculated in leaf litter composting experiment. Compost
quality was defined by decrease C : N ratio [4].
First phase, leaf litters were collected from Biological
Forest and Biology Field Station Gadjah Mada University,
Indonesia. Leaf litter colllected from ten different plot, use
Random Sampling method. For each plot, collected ± 500 g
leaf litter, then accumulated in the holes in the ground, with
the length, width, and depth, 0.75 m x 0.50 m x 0.30 m
(Figure. 1). Composting leaf litter naturally left for 20 days to
allow for natural decomposition process. Ten gram of the leaf
litter compos was asceptically introduced into 90 ml of sterile
distilled water and shaken vigorously. Subsequently serial
dillution suspension was plated onto selective medium.
Abstract— Utilization of leaf litter are still rare. Leaf litter only
accumulate or even burned. Burning waste can pollute the
environment. Use cellulolytic microorganisms, leaf litter can be
processed into compost that has economic value. The objective of
the research are to obtain suitable microorganism use for composting,
particularly for leaf litters composting. The method used in this study
is the selective isolation of cellulolytic bacteria and fungi litter at
both locations were grown on selective medium CMC, avicel, xylan,
and tannin, then the best selected isolates were inoculated in leaf litter
composting experiment. A total of 72 isolates microorganism were
isolated, 28 belong to bacterial and 44 belong to fungal. Leaf itter
with the addition of inoculum of bacteria and molds showed a faster
process than the litter without inoculums, with the final C / N ratio of
19.56 accordance with the standars of the compos Asia trade.
Keywords—Cellulolytic microorganism, Leaf litter, Composting.
I.
INTRODUCTION
U
TILIZATION accumulated leaf litters, such as in the
yard, sidewalks, and lawn are still rare, moreover adds to
the overall problem of municipial waste [1]. Leaf litters
are often accumulated piled up, discarded indiscriminately, or
set on fire [2]. Burning leaf litters can pollute the environment.
Leaf litters could be turned from liabilities into compos.
Leaf litters accumulated are potential substrate for compos
that has economic value. Composting of leaf litter need
specific microorganism because of leaf litter complex
composition. Leaf litter consisted of cellulosic material
including cellulose, hemicelluloses, and lignin that not easily
degrade [3]. Only a few microorgnisms are able to degrade.
Cellulolytic bacteria and cellulolytic mold which utilize
cellulosic materials for their energy source could be exploited
for the conversion of leaf litters into compos.
The availability of big amount of leaf litters as cellulosic
materials in Indonesia underlines the need to explore the
potentials of the cellulolytic bacteria and cellulolytic mold for
the transformation of these wastes into useful products like
compos.
The objective of the research are to obtain suitable
microorganism use for composting, particularly for leaf litters
Fig.1. Composting Hole
Isolation of cellulolytic bacteria from the leaf litter using a
modification selective medium described by [5]. The medium
contained the following per litre composition :0,8g K 2 HPO 4 ,
0,2g KH 2 PO 4 ; 0,2g MgSO 4 .7H 2 0 ; 0.2g NaCl ; 1 g NaNO3
; 0,01g CaCO 3 ; 0,5g yeast extract ; 20g agar and 10g
cellulosic materials (CMC, Avicel) for isolation cellulolytic
bacteria or Hemicellulosic material (Xylan) for isolation
hemicellulolytic bacteria. Isolation of cellulolytic fungi from
Ardhiani Kurnia Hidayanti, Faculty of Biology, Gadjah Mada University,
Yogyakarta, Indonesia ; phone : 0274 580839 ; fax : 0274 580589 ; (e-mail:
ardhiani.kurnia.h@mail.ugm.ac.id ).
Nungki Amalia Puspa Kirana, Faculty of Biology, Gadjah Mada
University,Yogyakarta,Indonesia(e-mail:crystalpalace3@gmail.com).
Endang Sutariningsih Soetarto, , Professor at Faculty of Biology, Gadjah
Mada University, Yogyakarta, Indonesia (e-mail: annisah-endang@ugm.ac.id
).
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3rd International Conference on Chemical, Biological and Environment Sciences (ICCEBS'2013) January 8-9, 2013 Kuala Lumpur (Malaysia)
the leaf litter using a modification selective medium described
by [2]. The medium contained per litre : 2,13 g Na 2 HPO 4 ; 1,3
g KH 2 PO 4 ; 0,5g NH 4 Cl ; 0,2g MgSO 4 .7H 2 O ; 20g Agar ;10g
cellulosic materials (CMC, Avicel) for isolation cellulolytic
bacteria, Hemicellulosic material (Xylan) for isolation
hemicellulolytic bacteria, and 5g lignin material (Tannin) [6].
Pure cultures of bacteria and mold were grown in selective
medium CMC (Carboxil Methil Cellulose), Avicel and Xylan
for test the ability of cellulose degradation. Then incubated for
2 days for bacteria and 7 days for mold in room temperature.
Colonies that grew then washed with a solution of Congo red
[7] for 10 minutes and rinsed with 1% NaCl. Visible colony
growth from colony formation and the presence of a clear
zone is the result of degradation of the CMC, Avicel and
Xylan by isolate bacteria / mold. Cellulolysis was assessed by
cellulolytic index by measuring the diameter of the clearing
zone around the outside of colony [8].
Selected bacteria and mold which have the biggest
cellulolytic index were characterization based on colony
morphology, the morphology of cells, and the biochemical and
physiological properties [9]-[10]. The date match observations
using Bergey's Manual of Determinative Bacteriology.
Identification of bacteria also using the BD Phoenix with 46
test kits based on biochemistry kit test.
Identification of fungi carried by observing morphological
characters both macroscopically and microscopically.
Observed macroscopic characters include color and surface
The data were identified using molds book [11].
Second phase, composting experiment with additional
selected microorganisms. Leaf litter collected from each study
site. Each treatment consisted of 2.5 kg leaf litter and was
given additional source of N form bird dung and goat dung (1
:0,04) [12]. Control is leaf litters without inoculation of
microorganisms, treatments were inoculated 1-2% inokulum
[13]-[14], contain 105-106 CFU/ml. First treatments were
inoculated with selected bacterial culture. Second treatment
was inoculated with a culture of selected fungi. The third
treatment was inoculated with selected bacterial and selected
molds.
Analysis of the C / N ratio was measured by determining
total nitrogen (Kjehdal) and determination of organic matter
(Walkey-Black) [15] and then determined the ratio of C / N
compost by the formula:
The ratio of C / N: Determination of Organic Materials
Total Nitrogen Determination
Analysis of levels of C / N, tested at the Soil Laboratory,
Faculty of Agriculture, Gadjah Mada University.
Fig. 2. Halozone around cellulolytic colony microorganism
CAR 4B is the best cellulolytic bacterial isolate which has
the bargest clearing zone and the biggest cellulolytic index
4.03±1.7 d. Cellulose in the medium induces the synthesis of
cellulase by bacteria. When the medium in the vicinity of
colonies of cellulolytic microorganism added reagents Congo
Red, the area does not provide a solid red color but showed
clear zone. Similarly, the bacterial isolates 48B XSS is the
best xylanolytic bacteria with xylanolytic index 3.3±0.47 c.
Mold isolates CK4 and AK51 are the best cellulolytic mold
which has cellulolytic index 4,92 and 3,20. XK15 is the best
xylanolytic mold with xylanolytic index 2,00, and TK70 is the
best lignolytic mold which has lignolytic index 1,38 .
Addition of inoculum in the litter accelerate the composting
process. From the results of this study shows that the addition
of inoculum microorganisms in the leaf litter showed a higher
temperature when compared with the control without the
addition of microorganisms. Initial temperature was 26 after
two weeks of composting litter temperature increased with the
addition of inoculum in the range 33-35 ° C. Temperature of
30-60 ° C is the optimum temperature during the composting
process takes place [16]. After almost one month of
composting, the temperature back down, indicating that the
litter has become compost.
pH during composting are in a neutral range from 6.5 to 7.5,
it is in line with the opinion of [16] which states that the
compost has a neutral pH. Humidity litter on days 0
composting by 36% after one month of composting, litter with
the addition of inoculum microorganisms have the humidity
above 50% humidity is common compost moisture, while the
control without the addition of microorganisms, after one
month of composting only have 29% humidity.
Color composting litter after one month with the addition of
inoculum showed differences when compared with the initial
litter composting (Figure. 4.) . At the beginning of composting
litter still green and yellowish brown, after the end of the
composting color changes to dark brown. Changes in physical
properties are the result of the decomposition process by
microbes, it also proves that the composted material (grass)
leaf chlorophyll loss (chlorophyll). It is also caused by
microbial activity that produces CO 2 and water.
Cellulolytic bacteria and fungi are microorganisms can
break down cellulose in litter in to a simpler compounds [17].
The process begins with the colonization of litter
decomposition by microorganisms such as bacteria and molds
that can decompose leaf litter through enzymatic mechanisms.
These microorganisms secrete enzymes that destroy molecules
of complex organic molecules such as cellulose from leaf
litter. Several enzymes are involved in the overhaul of organic
materials such as cellulase and ligninase.
A total of 72 isolates microorganism were isolated, 28
belong to bacterial and 44 belong to fungal.Cellulolytic
bacterial and cellulolytic mold isolates showing clear zone
around bacterial colonies (Figure 2.). Clear zone produced in
the presence of the D-cellobiose and showed that cellulose in
the medium has been hydrolyzed into simpler molecules by
cellulase enzymes produced by microorganism [3] .
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3rd International Conference on Chemical, Biological and Environment Sciences (ICCEBS'2013) January 8-9, 2013 Kuala Lumpur (Malaysia)
C / N ratio is one indicator of compost maturity.
Microorganisms break down the compound C as an energy
source and using N for protein synthesis. During composting
litter changes carbohydrates, cellulose, hemicellulose, fats and
waxes into CO 2 and water, as well as changes in the protein to
ammonia, CO 2 and water. This reduces the levels of
carbohydrates which are compounds C and increase soluble N
compounds in the form of ammonia, so the C / N ratio of litter
dropped close ratio C / N soil. On day 0 composting, C / N
ratio was 28.33, after composting for 36 days, a significant
decrease in leaf litter with the addition of microorganisms
(Figure 3).
Natural composting process takes a relatively long time. In
general, the composting process may take up to 8 week or 2-3
months even reached 6-12 months. But with the addition of
inoculum microorganisms in this research, composting takes
only 4-5 week. It is proved that microorganisms isolated from
Biological Forest and Biology Field Station Gadjah Mada
University, Indonesia, effective spur maturity litter into
compost in a shorter time than controls and reduced the
percentage of C / N ratio more quickly. Decline in the value of
C / N ratio is due to the inoculation of microorganisms in the
composting process and the microorganism will stimulate
microbiological processes during composting progress.
Fig. 3. C/N Ratio between varian treatment and control
Fresh leaf litter had a C : N ratio of 28.33. After
composting, the compos with bacterial inoculated gave a C :
N ratio 20.43, reducing the C : N rat by 16.20 % relative to the
starting material. The compos with mold inoculated gave a C
: N ratio 21.79, reducing the C : N rat by 13.04 %. The
compos with mold and bacterial inoculated produced an even
lower C:N ratio of
19.56, a 18.31% reduction. The
uninoculated treatment gave a C : N ratio of 26.45, not
significant reducing the C : N rat by 3.43%. Compos with
mold and bacterial inoculated showed the lowest C:N ratio,
bacterial and mold enzymes act synergistically to catalyse the
hydrolysis of cellulose [18] and more optimal activity in
degrading compost. The results of the final C / N ratio of
composting with the addition of mold and bacteria inoculums
qualified accordance with the standars of the compos Asia
trade defined by Food and Fertilizer Technology Center
(FFTC),C / N ratio < 20.
Plant litter is a source of carbon and nitrogen that can be a
substrate for microorganisms living. Various microorganisms
have the ability to decompose litter. Decomposition is the
process of decomposition and separation of organic materials
broken into sections. Decomposition of organic matter
occurred in the composting process by the addition of
inoculum of microorganisms and without the addition of
inoculum of microorganisms, but with the addition of
composting microorganisms inoculum compost maturation
process accelerated by microorganisms. Microorganisms play
an important role as an activator in the compost maturation
process.
Fig. 4. Comparative product leaf litter composting after fourth week
Characterization and Identification of Selected Isolates
based on morphology colony CAR 4B isolates of bacterial
colonies grown on agar plate has a rhizoid shaped, the colony
edges lacerate / ramose. These colonies creamy light brown
and opaque. While XSS 48B isolate bacterial colonies grown
on agar plate has a rhizoid shaped rhizoid, with lobate edge of
the colony. These colonies are cream colored and
opaque.Based on the morphology cell, bacterial isolates CAR
4B and 48B XSS are rod shape gram-positive bacteria.
The results of the BD Phoenix identification based on 46
biochemical tests, showed that the CAR 4B isolates, isolated
from the litter in the Biological Garden, has characteristics
similar to Bacillus subtilis. While XSS 48B isolates were
isolated from the litter in Biology Field Station, has
characteristics similar to Bacillus subtilis.
The results of mold characterization show that an isolate
CK4 similar to Basidiomycetes, AK51 similar to Pennicillium,
whereas isolates AK 15 and TK 70 similar to Aspergillus.
ACKNOWLEDGMENTS
The research was supported by IMHERE Grant Research,
Faculty of Biology, Gadjah Mada University.
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3rd International Conference on Chemical, Biological and Environment Sciences (ICCEBS'2013) January 8-9, 2013 Kuala Lumpur (Malaysia)
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