IJERD – International Journal of Environmental and Rural Development (2015) 6-1

Research article
erdLong-Term Effects of Spent Wash Liquor on Soil Bacterial
Population in Sugar Cane Production Systems
PHRUEKSA LAWONGSA*
Agricultural Biotechnology Research Center for Sustainable Economy: (ABRCSE),
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Email: phrula@kku.ac.th

PIROMYA MANEEWONG
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

NARUEMON NAKSRI
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

TIPPAWAN PROMCHART
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

SUTEE WONGMANEEPRATEEP
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

CHULEEMAS BOONTHAI IWAI
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

MONGKON TA-OUN
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Received 15 December 2014

Accepted 1 April 2015(*Corresponding Author)

Abstract Spent wash liquor (or Distillery Slop) is an agro-industrial wastes generated
during alcohol distillery production. Its application to the soil for agriculture is highly
beneficial due to rich in plant nutrients, organic carbon, less toxic and easily amenable formicroorganisms. An advantage is made to direct apply the spent wash liquor as asupplement fertilizer to enhance crop yield in agricultural field. Therefore, long-term effectsof spent wash liquor on soil bacterial population in agricultural crop production system isstill poorly understood. In this research, the effect of long-term application of spent washliquor on bacterial decomposers was investigated in sugar cane production systems in Nam
Phong watershed. Eight treatments in this research including soil with no spent wash liquor
applied (control), soil applied with spent wash liquor for 1, 2, 3, 4, 5, 7, and 8 years wereconducted. The results showed that bacterial population at 1 year after application was
significantly higher than those from control soil then, the population declined until 8 years
after application the population can reach up to 2.04 x 106 CFU/g soil. This study suggested
that application of spent wash liquor can affect the bacterial decomposer under sugar canefield. However, the application of spent wash liquor need to be tested in longer time underfarmer’s field conditions before further recommendation can be given.
Keywords spent wash liquor, sugar cane, bacterial population

INTRODUCTION
Nam Phong watershed is the most important watershed in the upper northeast region of Thailand.
Its catchment covers approximately 1,518,900 ha, extending to the five provinces of Chaiyaphum,
Khon Kaen, Loei, Nong Bua Lumphu, and Petchaboon (Sthiannopkao et al., 2007). Nowadays,
Nam Phong watershed is threatened by a variety of human activities. Besides problem of soil
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erosion from agricultural areas around Nam Phong watershed, sediments and wastewater fromindustrial areas consist of large and medium scale factories such as sugar mill, distillery mill,
paperboard mill, tapioca mill are also become a pollutant in the watershed.
Removing pollutants from wastewater by wastewater treatment is the preliminary way toremediate water and reduce soils contamination before discharging it into the river and watershed.
Among wastewater from industrial areas, spent wash liquor, agro-industrial wastes generated
during alcohol distillery production, becomes the most promising wastewater using for agricultural
production due to it contains high concentration of organic load, high amount of nutrients such as
nitrogen, phosphorous, potassium, sulphur and a large amount of micronutrients (Suganya andRajannan, 2009). There have been many studies done investigation the effect of spent wash liquor
on plant growth and yield (Rath et al., 2011; Suganya and Rajannan, 2009). However, there islimited knowledge on how spent wash liquor can shape the abundance of bacterial communities inagricultural area. Therefore, this study was designed to investigate how long-term spent wash
liquor affected the bacterial populations as well as soil properties for successful application of spentwash liquor under sugar cane production systems to reduce chemical fertilizer application and also
recycle treated wastewater for agricultural use.
OBJECTIVE
The purpose of this study was to investigate the influence of spent wash liquor on the bacterial
population in sugar cane production system in Nam Phong watershed.
METHODOLOGY
Study Sites and Soil Sampling
Study sites were selected from Nam Phong watershed, Nam Phong district, Khon Kean province
between latitude 16°42’ N to 16°48’ N and longitude 102°53’ E to 102°57’ E. The soil ischaracterized as a Chakkarat soil series. Soil samples were collected from sugar cane plantation
which cultivated sugar cane cultivar K92 on soil depth 0-15 cm. Year of the experiment was in2013. Eight treatments were used in this research, including soil with no spent wash liquor applied(control), soil applied with spent wash liquor for 1 year (1Y), soil applied with spent wash liquorconsecutively for 2 years (2Y), soil applied with spent wash liquor consecutively for 3 years (3Y),soil applied with spent wash liquor consecutively for 4 years (4Y), soil applied with spent wash
liquor consecutively for 5 years (5Y), soil applied with spent wash liquor consecutively for 7 years(7Y) and soil applied with spent wash liquor consecutively for 8 years (8Y). The spent wash liquor
was used for pre-sowing irrigation at rates of 6.4 m3/ha (40 m3/1600 m2).
Analysis of Chemical Properties of Spent Wash Liquor
Spent wash liquor characteristic analyses were done by studying the chemical properties of spentwash liquor (Table 1). Chemical properties studied include pH, electric conductivity (EC), totalalkalinity (as CaCO3), total dissolved solids (TDS), total solid, total suspended solids (TSS),
biochemical oxygen demand (BOD), chemical oxygen demand (COD), dissolved oxygen (DO) andsalinity were investigated by APHA standard methods (APHA, 1995). Organic matter was studied
by wet oxidation method (Walkley and Black, 1934), total nitrogen (as N) by micro Kjedahlmethod (Bremner, 1960), total phosphorus (as P2O5) by Bray II and colorimetric method (Bray andKurtz, 1945), total potash (K2O) by extracting with ammonium acetate 1N, pH 7.0 and measured
by flame photometer.
Soil Analyses
Soil studies were conducted after spent wash liquor was used for pre-sowing irrigation 6 months byⒸ ISERD

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IJERD – International Journal of Environmental and Rural Development (2015) 6-1

digging for three replications in each treatments of sugar cane production system. Soilcharacteristic analyses were done by studying the physical and chemical properties of the soil
(Table 2). Physical properties studied include soil moisture and bulk density through the core
method. Particle-size distribution and soil texture by hydrometer method. Chemical properties
studied include soil reaction by pH meter in ratio of 1:2.5 with water, EC by EC meter, cationexchange capacity (CEC) by the 1.0 M ammonium acetate (NH4OAC) method (Chapman, 1965),
total nitrogen (Total N) by micro Kjedahl method (Bremner, 1960), available phosphorus
concentration by colorimetric method (Bray and Kurtz, 1945), exchangeable potassium levelmeasured by flame photometer, organic carbon and organic matter in soil by wet oxidation method(Walkley and Black, 1934).
Bacterial Population Analysis
Bacterial population analysis was done by serial dilution plating technique on nutrient agar medium(NAM) (Dubey and Maheshwari, 2002). Plates with serial dilution plating were incubated at 28 °C
for 1-2 days. Bacterial populations were enumerated from the plates and expressed as CFU (colony
forming units) per gram soil.
Statistical Analysis
This experiment has been designed as the completely randomized design (CRD). F-test along withmethod of Duncan’s multiple range test (DMRT) was used to analyze the differences of theaverage on each experiment.
RESULTS AND DISCUSSION
Spent Wash Liquor Characteristic Analysis
The data of chemical properties of spent wash liquor are shown in Table 1. The results showed thatspent wash liquor used in this study were rich in organic matter and available certain nutrients. In
addition, spent wash liquor contained high pH and showed low values of biochemical oxygen
demand (BOD) and chemical oxygen demand (COD) suggesting that using of spent wash liquor issafe for agricultural application.
Table 1 Chemical properties of spent wash liquor
Spent wash liquor
pH
Electric Conductivity; EC
Total Nitrogen (as N)
Total Phosphorus (as P 2O5)
Total Potash (K2O)
Total Alkalinity (as CaCO3)
Total Dissolved Solids (TDS)
Total Solid
Total Suspended Solids (TSS)
Organic matter (OM)
Biochemical Oxygen Demand (BOD)
Chemical Oxygen Demand (COD)
Dissolved oxygen (DO)Salinity

Results
7.83
6.89
0.11
0.01
0.36
2647.19
10503.33
12900.00
373.330.58<5450.33
3.44

UnitdS/m%%%mg/L
mg/L
mg/L
mg/L
%mg/Lmg/L
mg/L
ppt

Soil Analysis
The data of physical and chemical properties of soil are shown in Table 2. Being very rich inorganic matters, the utilization of spent wash liquor in agricultural fields creates organicⒸ ISERD

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fertilization in the soil which raises the pH of the soil, increases availability of soil nutrients andcapability to retain water and also improves the physical structure of soil. The results of nutrient
contents analysis showed that application of spent wash liquor into soil significantly increased theexchangeable potassium content in the soil. Likewise, the previous report found that the applicationof spent wash liquor into Sansai soil series significantly increased the available potassium content
in the paddy field (Chatchaisiri et al., 2003).
Table 2 Physical and chemical properties of soilTreatments
FCV
Soil
test (%)
properties
Control
1Y
2Y
3Y
4Y
5Y
7Y
8Y
Organic
matter;0.603a0.333e0.473c0.358e0.337e0.535b
0.515b0.440d*21.64
OM (%)
Total N (%)
0.027a0.013d0.02c
0.014d
0.013d
0.023b
0.023b0.018c*
29.14
Available Pacecbf
gd
48.64
36.19
24.96
36.53
42.3
20.37
13.44
33.58
*
35.26(mg/kg)
Exchangeable K
79.10c80.48c38.85e72.29d39.32e116.34a81.87c108.91b*35.20(mg/kg)
Organic
carbon;0.344a0.194e0.256c0.215d0.198e0.309b
0.306b
0.263c*20.85
OC (%)
Soil pH
4.85d
5.50b
5.30c
6.41a
5.63b
5.69b
4.85d
4.74d
*
10.19
Electricalconductivity;
0.042a0.029d
0.015g0.020f0.039b
0.035c0.039b
0.023e*32.43
EC (dS/m)Cationexchange
capacity;
3.371b
3.627b
5.051b
3.559b
4.612b
1.190c8.088a1.272c*
57.59
CEC(cmol/kg)
Bulk density
0.890bc
0.960a0.910b
0.910b0.950a0.950a0.860c0.855c*4.59(g/cm3)
Soilmoisture
3.860d4.420b3.300f
3.550e4.150c5.880a3.415ef
4.265bc*
19.48(%)
Soil texture
sand
sand
sand
sand
sand
sand
sand
sand
Treatment: soil with no spent wash liquor applied (control), soil applied with spent wash liquor for 1 year (1Y), soilapplied with spent wash liquor consecutively for 2 years (2Y), soil applied with spent wash liquor consecutively for 3years (3Y), soil applied with spent wash liquor consecutively for 4 years (4Y), soil applied with spent wash liquor
consecutively for 5 years (5Y), soil applied with spent wash liquor for consecutively 7 years (7Y) and soil applied withspent wash liquor consecutively for 8 years (8Y). In column, means followed by the same letter do not differ statisticallyfrom each other at pd0.05 according to DMRT test.

Effect of Spent Wash Liquor on Bacterial Population in Sugar Cane Production System
The highest total count of bacteria was found in soil sample that taken from first year application ofspent wash liquor for 2.11 x 106 cfu/g soil (Table 3). However, bacterial population tend todecrease when soil were applied with spent wash liquor consecutively for 2-7 years. Interestingly,
after 8 years consecutively application of spent wash liquor, result showed increasing in thebacterial count as compared to sugar cane production system without any spent wash liquor
application. Jintaridth et al., 2002 reported that application of spent wash liquor into soil showed
increasing in bacterial and fungal populations at the rate of 40 m3/1600 m2 compared to treatmentusing chemical fertilizer. Moreover, Thitakamol and Kaewpinthong (2004) also found that, at therice post-harvesting period, the number of bacteria increased at the application of 40 m3/1600 m2 ofspent wash liquor and the number of fungi was increased at the using rate of 20 m3/1600 m2. The
bacterial populations can change due to longer time of spent wash liquor application. The bacterial
populations may be affected by changing in soil moisture and some certain soil nutrient such aspotassium. The results revealed that significantly higher in soil moisture and exchangeable K valueⒸ ISERD

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IJERD – International Journal of Environmental and Rural Development (2015) 6-1

were found in soil applied with spent wash liquor for 1 year (1Y) and soil applied with spent washliquor consecutively for 8 years (8Y) (Table 2). These findings suggested that the growth andpopulation of soil microorganisms can be influenced by chemical and physical properties of the soil
and the availability of macro nutrient elements can limit bacterial population growth in a particularsoil ecosystem.
Table 3 Effect of spent wash liquor on bacterial population
Treatment
Control
1Y
2Y
3Y
4Y
5Y
7Y
8Y
F-testCV (%)

Bacterial count (cfu/g soil)
1.45 x 106 b
2.11 x 106 a
1.48 x 106 b
7.07 x 105 d
1.08 x 106 c
8.67 x 105 cd
7.73 x 105 d
2.04 x 106 a*40.97Treatment: soil with no spent wash liquor applied (control), soil applied with spent wash liquor for 1 year (1Y), soil
applied with spent wash liquor consecutively for 2 years (2Y), soil applied with spent wash liquor consecutively for 3years (3Y), soil applied with spent wash liquor consecutively for 4 years (4Y), soil applied with spent wash liquor
consecutively for 5 years (5Y), soil applied with spent wash liquor for consecutively 7 years (7Y) and soil applied withspent wash liquor consecutively for 8 years (8Y). In column, means followed by the same letter do not differ statisticallyfrom each other at pd0.05 according to DMRT test.

CONCLUSION
The study of long-term effects of spent wash liquor on soil bacterial population in sugar cane
production systems has shown that the highest total count of bacteria was found in soil sample thattaken from first year application of spent wash liquor and then bacterial population tend to decrease
when soil were applied with spent wash liquor consecutively for 2-7 years and raised up in soil
sample that taken from eight years consecutively application of spent wash liquor. This finding
suggested that bacterial populations may be affected by changing in soil physical and chemical
properties such as soil moisture and some certain soil nutrient such as potassium. Therefore, anessential soil element for plant growth influences the microbial population as these nutrient
elements are also needed for microbial growth and activity. However, the application of spent wash
liquor need to be tested in longer time under farmer’s field conditions before further
recommendation can be given.
ACKNOWLEDGEMENTS
This work was supported by research grants from Khon Kaen University; project no. 562402 and572702.
REFERENCES
APHA, AWWA, WPCF, 1995. Standard methods for the examination of water and wastewater, 16th Edition.
American Public Health Association, American Water Works Association and Water Pollution Control
Federation, Washington, D.C., USA.
Bray, R.H. and Kurtz, L.T. 1945. Determination of total, organic, and available forms of phosphorus in soils.
Soil Sci., 59, 39-45.
Bremner, J.M. 1960. Determination of nitrogen in soil by the Kjeldahl method. J. Agr. Sci., 55, 11-33.
Chapman, H.D. 1965. “Cation exchange capacity,” in Methods of soil analysis: chemical and microbial
properties. Black, C.A. Ed., vol. 9 of Agronomy Series, American Society of Agronomy. USA. 891-901.
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IJERD – International Journal of Environmental and Rural Development (2015) 6-1

Chatchaisiri, K., Chanchareonsook, J., Suwannarat, C. and Panichsukpatana, S. 2003. Utilization of paper
mulberry pulping waste water and distillery slop as sources of potassium for rice grown on Sansai soilseries. Proceedings of 41th Kasetsart University Annual Conference. Thailand. 26-33.
Dubey, R.C. and Maheshwari, D. K. 2002. Practical microbiology. S. Chand and Co., New Delhi, India.
Jintaridth, B., Kanjanathanaset, K., Inthasothi, T., Leaungvutiviroj, C. and Charoenchamratcheep, C. 2002.
Utilization of spent wash liquor for rice production in acid sulphate soils. Presentation: poster 17thWCSS, 14-21 August. Paper No. 2282.
Rath, P., Pradhan, G. and Misra, M.K. 2011. Effects of DSW (distillery spent wash) and fertilizer on growthand chlorophyll contents of sugar cane (Saccharum officinarum L.) plant. Recent Res. Sci. Technol., 3,169-176.
Suganya, K. and Rajannan, G. 2009. Effect of one time post-sown and pre-sown application of distillery
spent wash on the growth and yield of maize crop. Bot. Res. Inter., 2, 288-294.
Sthiannopkao, S. Takizawa, S. and Wirojanagud, W. 2007. Assessment of seasonal variations of surface
water quality in the Phong Watershed, Thailand. Thammasat Int. J. Sci. Technol., 12, 36-43.
Thitakamol, T. and Kaewpinthong, S. 2004. Utilization of distillery slop for the rice production in Khon
Kaen province. Ramkhamhaeng University Research Journal, 7 (Special Issue), 132-151.
Walkley, A, Black, I. A. 1934. An examination of the Degtjareff method for determining soil organic matter,and a proposed modification of the chromic acid titration method. Soil Sci., 37, 29-38.

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