Chemical, Physical and Microbiological Changes during Composting of the Water Hyacinth
An investigation of the physical, chemical and microbial population changes that occurred during the composting of water hyacinth was carried out. After 11 weeks of composting, the compost turned black, had decomposed and had no smell. The pH was 7 and the highest temperature reached, of 40°C occurred in the first week. The initial carbon/nitrogen ratio was 17.61 and this increased to 18.12 by the end of the composting. The coliform population declined greatly from 8.11 to 5.85 MPN (log)/g and fecal coliforms and Escherichia coli were not detected in the final product. Bacteria were the dominant microbes in the compost followed by actinomycetes and fungi. Mesophillic microorganisms were present in higher numbers than thermophillic microorganisms throughout the composting. The highest cellulase and xylanase activities in the compost of 6.67 and 10.24 U/kg DW, respectively were detected in the second week which was related to the temperature. Bacillus sp. strain B4 was isolated and investigated for cellulase and xylanase using agro-industrial residues as substrates during Solid-State Fermentation (SSF) processes. Corncob and rice straw were good substrates for the production of the enzymes with a maximum cellulase of 1.19 U/gDW and xylanase activity of 2.54 U/g DW, respectively. The activities of both enzymes were stable and maximum at 50°C. This study indicated that agro-industrial residues should be mixed with water hyacinth for composting to facilitate the development of a thermophilic phase during the composting process and to improve the product. Bacillus sp. strain B4 can be used as a starter strain.
Received: June 25, 2010;
Accepted: August 16, 2010;
Published: September 24, 2010
The water hyacinth (Eichhornia crassipes), a native aquatic weed of
Brazil and possibly other central South American countries (Center,
1994) is an invasive aquatic macrophyte with a potential for particularly
rapid growth and dispersal (Cook, 1990). It has been spread
around the world by humans (Gopal, 1987) and can cause
considerable damage to local environments, human health and economic development
(Fernandez et al., 1990; Epstein,
1998). Lately the water hyacinth has invaded many tropical lakes rivers
and canals especially in Thailand and has had a big impact on local communities.
Its rapid growth has reduced the supply of clean potable water, caused difficulties
in water extraction, blocked irrigation canals, increased transportation costs,
reduced fish catches, decreased available landing sites and depleted the major
nutrients present in normal non potable water sources (Gunnarsson
and Petersen, 2007; Malik, 2007).
In Thailand, with the favorable conditions for growth, water hyacinth causes
major problems in many irrigation systems and because of the high cost of removal
and its rapid growth this weed mostly remains unharvested and unutilized. Although
water hyacinth is considered to be one of the worlds most environmentally
unfriendly aquatic plants, there have been many efforts to try to devise useful
applications such as for use as a raw material for better value products including
paper, wine, hand crafts and biogas. One of the easiest ways to utilize water
hyacinth is to subject it to composting. Water hyacinth compost has been shown
to have positive effects on plant growth (Sharma and Mittra,
1990; Abdel-Sabor and Abo El-Seoud, 1996; Chukwuka
and Omotayo, 2009). The use of composted organic wastes as amendments to
improve soil organic matter levels and long term soil fertility and productivity
is gaining importance. Compost is one of the key factors in producing organic
foodstuffs. Haug (1980) defined composting as the biological
decomposition and stabilization of organic substrates under conditions that
allow development of thermophilic temperatures as a result of biologically produced
heat, with a final product sufficiently stable for storage and application to
land without any adverse environmental effect. It has always been a widely used
method for disposal of organic wastes (Goyal et al.,
2005). Mesophilic and thermophilic microorganisms are involved in the composting
and their succession is important for the effective management of the composting
process (Beffa et al., 1996; Ishii
et al., 2000). The heat generated during composting helps to destroy
potential pathogens and controls the relationships between microorganisms (Feinstein
et al., 1986; Schlegel and Jannasch, 1992).
Different hydrolytic enzymes are released by the different microorganisms and
they cause depolymerization of the many different waste organic polymers (Kandeler
et al., 1999; Marx et al., 2001).
Important enzymes involved in the composting process include different cellulases
and xylanases, other glycosidases, phosphatases and proteases. High levels of
cellulase and xylanase activities have been detected throughout the active phase
of composting (Cunha Queda et al., 2002; Mondini
et al., 2004) and many of these enzymes are thermostable.
Composts prepared from different organic wastes differ in their quality and
stability. This mainly depends upon the composition of the raw material used
for the composting process (Gaur and Singh, 1995; Ranalli
et al., 2001). Compost quality is closely related to its stability
and maturity. As many different chemical and biological changes occur during
composting, the choices of raw materials and the composting method have made
it difficult to agree on methods for the practical assessment of maturity (Itavaara
et al., 2002; Benito et al., 2003;
Wang et al., 2004). Various parameters have been
used to assess the quality and maturity of composts such as CO2 production,
the C:N ratio of the finished product, presence of potential pathogens such
as coliform bacteria, moisture, temperature and pH of the finished compost (Garcia
et al., 1992; Huang et al., 2001;
Wu and Ma, 2002; Al-Turki, 2010).
Water hyacinth compost has been used widely in Thailand but there has been little study in terms of its microbiological changes. This present study was conducted to monitor the physical, chemical and microbiological changes during composting of the water hyacinth and also to select bacteria with the potential to produce high levels of cellulase or xylanase activities.
MATERIALS AND METHODS
Composting and sampling protocol: Water hyacinth was collected from the Pakphanang River, Nakhonsithammarat in January, 2008. It was dried by sunlight for 2 weeks and then mixed with cattle dung and coconut shell (9:3:1). The material was shaped into 2m x 2m x 70 cm pile and allowed to decompose for a period of 3 months. During composting the moisture content was measured every week and kept at a level of 60%. The compost was turned every 2 weeks. Compost samples were drawn from three different points of the pile every week to investigate for microorganisms, pH, moisture content and enzyme activities. All analysis was performed in triplicate.
Physio-chemical and biological analysis: Total carbon was analyzed by
dynamic flash combustion and total nitrogen was estimated by N/protein analysis
(Horwitz, 1997). The pH of the compost was determined
in distilled water with a 1:10 (w/v) compost: water ratio. The moisture content
was measured by drying to constant weight in an oven at 105°C. Changes in
the temperature of the compost pile were recorded using a mercury thermometer.
The mean of the pile temperature at two monitoring points is reported. Cellulase
and xylanase activities were estimated using carboxymethyl cellulose and xylan
as substrates respectively. The reducing sugars released by enzymes were determined
by the method of Nelson-Somogyi (Somogyi, 1952). One
unit of enzyme activity was defined as the amount of enzyme required to liberate
1 μmole of glucose per minute and was expressed as U.
Coliforms, fecal coliforms and E. coli present in the first and final
weeks of composting were determined by the most probable number method (MPN)
(APHA, AWWA, WPCF, 2005). The number of bacteria, fungi
and actinomycete were counted by the diluted plate count method on TSA (tryptic
soy agar), Rose bengal medium and actinomycete isolation agar, respectively.
The incubation temperature was 35°C for mesophilic and 50°C for thermophilic
microorganisms. Random bacterial colonies on TSA were selected and tested for
cellulase and xylanase enzyme production on CMC and xylan agar respectively.
In order to detect cellulase or xylanase activity, plates were flooded with
0.1% (w/v) Congo red solution for 30 min and then rinsed several times with
1 M NaCl (Chen et al., 2004). This procedure
revealed distinct hydrolysis regions. Isolates producing a high degree of hydrolysis
were then further investigated for enzyme production and identity.
Enzyme assay: Enzyme production was investigated by a solid state fermentation
using different agricultural wastes including water hyacinth, coconut shells,
rice straw and corncob. Each substrate was cut into 1 cm pieces and oven-dried.
Seventy grams of each substrate was mixed with 150 mL of Bergs mineral
salts medium (Berg et al., 1972) and the moisture
content was adjusted to 70% by adding distilled water. Substrates were autoclaved
before use. Bacteria were grown in Bergs mineral salts medium at 35°C
for 24 h. Cell suspensions were adjusted to the 0.5 McFarland standard and 1
mL of culture was inoculated into substrate. Cultures were incubated at 35°C
for 13 days. Cellulase and xylanase were assayed everyday.
Effect of temperature on activity and stability of enzymes: Bacterial isolates were grown with water hyacinth mixed with coconut shells (7:1) and 150 mL of Bergs mineral salts medium. The moisture content was adjusted to 70%. After 6 days of incubation at 35°C, enzyme activities were determined at 40, 50, 60, 65, 70 and 80°C to study the effect of temperature on enzyme activity. For the thermal stability test, the enzyme extract was incubated with substrates at 50, 60, 65, 70, 80 and 90°C for 30 min before determining enzyme activity as mentioned.
Bacterial identification: Isolates with high ability of enzyme production were identified by a partial 16s rDNA analysis and their morphological and physiological properties.
RESULTS AND DISCUSSION
Changes in temperature, pH and moisture at various stages of decomposition
are shown in Fig. 1a-c; the pH of the compost
remained stable at 7 during the 11 weeks of the process. An initial temperature
of 28°C was recorded and the highest temperature, of up to 40°C, was
observed at 7 days of composting and then declined gradually. It has been previously
reported that the increase of temperature in the early phase of composting may
be the result of a thermophilic phase of composting. The temperature rises as
a consequence of the rapid breakdown of organic matter, mostly readily available
substrates rich in carbon, by microbial metabolism (Kutzner,
2001). Although studies of Inbar et al. (1993)
and Tiquia (2002) suggest that in the thermophilic phase
the temperature could rise up to 60-70°C, in this study an increase of only
to 40°C was observed. This could be attributed to the differences of material
used and the composting process. The moisture content was high throughout the
process at 80% in the first week and 70% in the final week.
The initial C: N ratio of the water hyacinth compost was low at 17.61. An unexpected
increase in C: N ratio to 18.12 was found at the end of composting (Table
1). The initial content of carbon however gradually decreased as the decomposition
progressed. The decrease in total N content was due to conversion of organic
nitrogen to ammonia and the subsequent loss of ammonia. This of course depends
upon the type of material being composted and its C:N ratio. The increase of
C:N ratio may be due to a low initial C:N ratio that results in more N losses
than in high C:N ratio wastes (Sanchez-Monedero et al.,
2001). This agreed with the findings of the study done by Goyal
et al. (2005) that an initial low C/N ratio of water hyacinth led
to a nitrogen loss compared to other substrates with high C/N ratio.
It has been reported that a lower C:N formulation exhibited a slower increase
in temperature and was an indication of poorer decomposition (Huang
et al., 2004; Erickson et al., 2009).
C/N is among one of the important factors affecting compost quality (Michel
et al., 1996).
||Changes in (a) Percentage moisture, (b) pH and (c) Temperature
|| Carbon and nitrogen content, C:N ratio and the amount of
coliform bacteria and Escherichia coli of the compost
|nd*: Not detected
It is recommended to start composting with C/N at 25-30 as this is considered
to be the optimum ratio (Huang et al., 2004).
Only a 10% carbon decrease was detected in this study compared to Atkinson
et al. (1996) who found about a 29% reduction during the composting
of poultry litter with saw dust. The loss was due to the release of carbon dioxide.
Even though the C:N ratio did increase, any value of below 20 can be considered
satisfactory for use in the field (Kutzner, 2001; Raut
et al., 2008).
The presence of pathogens in the compost was investigated by determining the numbers of coliforms, fecal coliforms and Escherichia coli at the beginning and the end of the process. Coliform bacteria reduced by more than 2 log MPN/g (Table 1). High amounts of fecal coliforms and E. coli present at the start with 7.04 and 6.7 log MPN/g, due to the use of cattle dung, were reduced to non detectable levels. Even though there is no criterion for an allowable number of pathogen in compost in Thailand, the determination of indicator microorganism is essential. In this study, we investigated coliforms, fecal coliforms and E. coli only as indicator organisms. Other pathogens may be less affected by biodegradation and could survive for much longer times. Other resistant pathogens must be considered with respect to handling safety.
Various hydrolytic enzymes control the rate at which various substrates are
degraded. Enzymes are the main indicators of various degradative processes (Tiquia
et al., 1996; Tiquia, 2002). Therefore the
changes in the activities of two important enzymes; cellulase and xylanase which
are responsible for hydrolysis of cellulose and xylan, respectively, were studied
to understand the degradation of organic wastes. The cellulase and xylanase
activity increased during decomposition and was maximum at 2 weeks (6.67 and
10.24 U/kg DW, respectively) followed by a decline (Fig. 2).
The results agree with those described by Goyal et al.
(2005) in that the cellulase activity increased during decomposition, reached
a maximum at 30 days and declined further at 60 and 90 days. The reports of
Raut et al. (2008) and Castaldi
et al. (2008) also showed that the production of hydrolytic enzymes
was maximized within 2 weeks of composting. These results indicate the importance
of the initial phase of composting. Because of the levels of xylanase and cellulase
in the compost they also most probably have important roles in the composting
processes of water hyacinth.
Microbial succession is known to play a key role in the composting process
and the appearance of some microorganisms reflects the quality of the maturing
compost (Ishii et al., 2000). The population
of mesophilic and thermophilic bacteria, fungi and actinomycetes at various
stages of the composting process was determined.
||Changes in cellulase and xylanase activity during composting
||Population of (a) Bacteria, (b) Actinomycetes and (c) Fungi
during composting; mesophiles and thermophiles
Bacteria were the dominant group throughout the process at more than 2 log
CFU g-1 higher than for fungi and actinomycetes. Mesophilic bacteria
and fungi were found to be at their highest numbers during the first week of
composting (Fig. 3a-c) with numbers of 8.6
and 5.7 log CFU g-1, respectively and then both declined.
Fungi have been reported to be an important group in the early phase of composting
(Klamer and Baath, 1998). The population of fungi was
highest at 4.4 log CFU g-1 in the first week with only slight changes
during the first 7 weeks and then a 1 log CFU g-1 decline was observed.
The thermophillic groups were lower than the mesophilic groups and changed only
slightly compared to the mesophilic groups. The lower numbers and changes in
the thermophilic microorganisms correlated with the low temperature of the compost.
This indicated that the poor decomposition resulted in low temperature rises
so there was little selection for any thermophilic microorganisms.
The development of mesophilic and thermophilic microorganisms during composting
is related to the mesophilic and thermophilic stages of the composting process
(Diaz-Ravina et al., 1989; Davis
et al., 1991; Ishii et al., 2000;
Riddech et al., 2002), thus as the maximum temperature
reached was only 40°C at day 7 of the composting there was no observed succession
from a mesophilic to a thermophilic microflora.
Isolation of bacteria with an ability to produce good levels of cellulase and
xylanase, led to the selection of isolate B4. The investigation of enzyme production
was performed using 4 different agricultural substrates, water hyacinth, coconut
shells, rice straws and corncob. All substrates produced xylanase activity that
was more than two fold higher than the cellulase. Cellulase was maximum at day
5 with 1.19 U/g DW from corncob while xylanase was highest at day 9 with 2.54
U/g DW from rice straws (Fig. 4a, b). Surprisingly,
water hyacinth produced the lowest activity for both enzymes. The reason for
this could be that water hyacinth has the lowest C/N ratio compared to other
substrates therefore not so much excess of carbon.
Both enzymes exhibited maximum activity (Fig. 5) and stability at 50°C (Fig. 6) and declined above this temperature although the activity of both enzymes was still at 80% at 60°C (Fig. 5). The cellulase and xylanase retained 20% of activity at 80°C. The stability of xylanase declined more rapidly when the temperature rose.
Agricultural wastes in the form of cellulose and xylan are the most abundant renewable resources in the biosphere and can be used in the production of valuable value added products. In this study, rice straws, corncob, coconut shells and water hyacinth have shown to be used as substrates to produce high amounts of enzymes. These results highlight the industrial potential of these substrates as possible raw materials for cellulase and xylanase enzyme production by microorganisms.
The isolate strain B4 was initially characterized to be a yellow-white colony,
Gram-positive, rod shaped and produced endospores. The resulting 16s rDNA partial
sequence were compared with those in the GenBank database using the BLAST program.
Results showed that B4 was a Bacillus sp. (Table 2).
||Time profile for cellulase (a) and xylanase (b) production
from strain B4 using 4 different substrates; water hyacinth, coconut shells,
rice straws and corncob at 35°C for 13 days
||Effect of temperature on the relative activity of cellulase
and xylanase from strain B4
||Effect of temperature on the stability of cellulase and xylanase
from strain B4
|| Identification results of strain B4
Water hyacinth is a potential unlimited resource for composting in Thailand. In this study the composting process was performed in the way that local people make their water hyacinth compost. We found some problems with their process. Since water hyacinth can readily absorb water, it is essential to first dry it and ensure that the moisture content at the start is not more than 70%. Chemical, physical and microbiological changes during composting of various organic wastes have indicated that there is a succession of microbial populations that depend on the temperature reached during the composting process. In this process there was no clear microbial succession because the maximum temperature attained was only 40°C. It is crucial in the first month of composting to control the right conditions for microorganisms. Most parameters do not change much after 6 weeks so the time for production can be significantly reduced. The water hyacinth compost used here starts with a low C/N ratio of 17.61 which is well below the optimum ratio of 25 to 30. This presumably is also the reason for the low maximum temperature as microbial metabolic rates are limited. However the final C:N ratio of 18.12 indicates that it might certainly be a good product to improve the fiber content of soil and this might offset its low N content. Additional agricultural wastes with very high organic carbon content added at the start should increase the C/N ratio. Rice straw, corncob and coconut shells are cheap residues that can be used as additional substrates for composting and also for increasing polysaccharide hydrolases such as cellulase and xylanase enzyme production and also temperatures to help with sterility. The isolate, Bacillus sp. B4, was shown to produce reasonable amounts of cellulase and xylanase activity. It will now be used as a starter strain for the composting process.
Financial support was provided by the research grant of the Faculty of Science, Prince of Songkla University. The authors are grateful to Dr. Brian Hodgson for his advice. We would like to thank the Marine and Coastal Resources Institute (MACORIN) for help with the composting process.
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