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Revista de la ciencia del suelo y nutrición vegetal

On-line version ISSN 0718-2791

R.C. Suelo Nutr. Veg. vol.10 no.2 Temuco  2010 

R.C. Suelo Nutr. Veg. 10(2): 158 -169 (2010)




Paula Aguilera1, Gabriela Briceño1, María de la Luz Mora1, Rolando Demanet2, Graciela Palma1*

1Scientific and Technological Bioresource Nucleus (BIOREN-UTRO), Universidad de La Frontera, Avenida Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile.
2Departamento de Producción Agropecuaria, Universidad de La Frontera, Avenida Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile. *Corresponding author:


The aim of this work was to establish the effect of the dose of liquid cow manure (LCM) on some chemical properties and biological activity in an Andisol. This study was carried out under laboratory conditions using an Andisol and LCM from a dairy farm located in the South of Chile. The dose of LCM were the equivalent to 0 (SO), 100 (S100), 200, (S200) and 300 (S300) thousand L ha-1. This amount is equivalent to regular agronomic LCM applications. The chemical characterization indicated that the LCM addition modified all the tested Andisol chemical properties. These begin to return to the initial conditions from 10 days on, with the exception of EC, K and DOC. The C-C02 evolution indicated that LCM incorporation at increasing dose enhanced the respiratory activity. Enzyme activity was greater in the amended soils than in SO. The results of this study indicated that the period of time necessary for the LCM amended soils returning to a similar behavior to SO is more than 10 days, with a strong correlation between the chemical properties of the amended soils and their biological activity, mainly C-C02 evolution.

Keywords: liquid amendment, biological activities, chemical analysis.



In recent years, the number of dairies, especially small scale dairies, in southern Chile has increased, and this growth has been accompanied by the intensification of production systems, with milking herd remaining in confinement during the majority of its productive period, fed with large quantities of concentrate, hay and fodder maize (Zea mays L.) silage (Aguilera et al, 2009).

The high feed requirements of the dairies result in the production of large volumes of liquid cow manure (LCM), which are a mixture of cattle urine, faeces, dairy washing water, rainwater and waste food. The LCM is a liquid amendment that contains less than 15 % of dry matter, containing organic matter with a high content in active functional groups, which also generates an increase in the microbial biomass and presents a high content of dissolved organic matter (Salazar, 2007; Briceño, 2008). The LCM has become an important resource, being recycled and incorporated into the production system. This situation is of great importance in southern Chile, since 80 % of the country's dairies are concentrated in this area (ODEPA, 2010). The application of the LCM to the soil is considered to be a practical solution due to the beneficial effects of its biological and physicochemical properties (Eghball et al, 2005; Wiederholt et al., 2005), such as increasing the soil organic matter content, contributing to nutrient recycling (Demanet et al., 1999), and increasing microbial activity by the direct addition of microorganisms (Balesdent et al, 2000; Chantigny et al, 2002), which together help to increase soil fertility due to LCM is an important source of macro and micronutrients (Aguilera, 1995).

Today, the LCM application is recommended in grassland and fodder maize. It has been shown, that a greater production of these crops is achieved by LCM application to the soil, especially in fodder maize used as a feed for milking herds (Demanet et al., 1999). However after LCM application has been observed that dense weed populations emerge and invade the fodder maize crop. Furthermore, there are weed species with a high capacity to capture the nutrients provided by LCM. Then these become invasive species which are difficult to control (Demanet, 1999).

At present, there is no uniform agronomic management of fodder maize in intensive dairy systems in southern Chile with respect to the LCM dose used, which exceed 300 thousand L ha-1, per year (Demanet et al, 1999; Salazar et al., 2003; Dumont, 2004).

Currently, the use of amendments originated in animal or agricultural waste has become a common worldwide agronomic practice, modifying chemical and biological soil properties (Perucci et al., 2000; Said-Pullicino et al., 2004; Briceño et al, 2007; Perucci et al, 2008). The aim of this study was to evaluate the application effect of increasing LCM dose on the chemical and biological properties in an Andisol. The Andisol under LCM application was analyzed for pH, organic matter (OM), electrical conductivity (EC), Nitrogen (N), phosphorus (P), potassium (K) and dissolved organic carbon (DOC). The biological activities evaluated were fluorescein diacetate (FDA) hydrolysis , respiratory activity (C-C02 evolution) and one soil enzyme activity associated to carbon cycling, β-glucosidase.



Soil and LCM

The Andisol used was a Los Lagos series, mesic, Durudands Typic (Andisol) (CIREN, 2003), with a sandy loam texture (500 g sand kg-1, 480 g silt kg-1, 20 g clay kg-1), taken from Fundo Santa Isabel, located in Futrono district (40° 03' 34"S; 72° 32' 46" W), Los Rios Region, Chile. The soil was extracted at 0 - 20 cm depth, sieved through 2 mm and air dried.

A single LCM sample was taken, obtained from an open air lagoon located in the area of the dairy farm. This lagoon was built with a cement base and lateral walls operating as an open system. The LCM application dose corresponded to 0, 100, 200 and 300 thousand L ha-1. In the laboratory, these doses corresponded to 70, 140 and 210 mL kg-1 in one application, assuming LCM distribution in the first 20 cm of depth and a soil density of 0.75 g mL-1.

Chemical LCM and Andisol analyses

LCM was analyzed following Peters et al. (2003) methodology. EC and pH were measured in the diluted LCM. For DOC analysis, the LCM was centrifuged, filtered, freeze-dried and analyzed as total organic carbon using a EURO EA 3000 Elemental Analyser with a Callidus software version 4.1. The experiment was done in duplicate. The obtained values were expressed in mg DOC L-1. Dry matter (DM) content was determined by water evaporation at 70°C until constant weight. The LCM characteristics were: DM 53.0 g L-1, EC 5.9 dS m-1, OM 71.1 mg kg-1, DOC 2,140 mg L-1, pH 7.5, and N 28.6 g kg. DOC in amended and non-amended Andisol was extracted using distilled water in a solid/liquid ratio of 1:10 (w/v) basis dry weight, using 25 g of soil and 250 mL of water. These samples were shaken, centrifuged, filtered and freeze-dried. The total organic carbon was analysed using the method indicated above. The Andisol was characterized as previously described by Briceño et al. (2008). The main soil characteristics were a 5.3 pH in water and OM 18.6 g 100 g-1.

Incubation of the amended Andisol

After LCM application, the different treatments were incubated for 1, 5, 10, 20, 30, 45 and 60 days in a dark room with controlled temperature at 20 ± 1 °C. The humidity content was adjusted and maintained by the addition of distilled water, whenever necessary. The amended Andisol were characterized chemically after the previously mentioned periods using the same method as for the non-amended Andisol (Table 1).

Biological analyses

Microbial respiration was determined by measuring C-C02 evolution according to the method described by Alef (1995), in which the C02 produced by the samples is captured by the NaOH solution and the remainder is titled with HC1 0.5 M. This vial containing alkali was removed, BaC12 was added in order to precipitate carbonates, and phenolphthalein was used as indicator. This vial containing alkali was replaced after each measurement. The data were expressed as C-C02 ug g-1 soil. The experiment was made in triplicate. The respiratory rate was fitted to a first order kinetic (Matus et al, 2009).

The measurement of FDA hydrolysis was performed using the methodology described by Adam and Duncan (2001). One gram of sample was extracted and placed in 50 mL centrifuging tubes. A buffer solution of potassium phosphate 7.6 pH was added and enzyme substrate fluorescein diacetate was added, after 10 min. The tubes were closed and incubated at 30 °C in an orbital incubator at 100 rpm for 20 min, at the end of which a solution of chloroform/methanol (2:1) was added to each tube. Then, tubes were then centrifuged for 20 min at 3,000 rpm. Finally, the fluorescein quantity was determined reading the supernatant in a spectrophotometer at a wavelength of 490 nm. Each evaluation was done in triplicate, with a blank and a control. The activity degree was evaluated on days 1, 3,5, 10, 20, 30, 45 and 60.

The data obtained by spectrophotometry were interpolated in a calibration curve using different fluorescein concentration, and then Lig fluorescein g-1 dry soil h-1 was calculated. The Andisol under LCM application incubated for 1, 3, 5, 10, 20, 30, 45 and 60 days were used for determining (β-glucosidase activity. The activity of this enzyme was evaluated using the methodology described by Eivazi and Tabatabai (1988).

Statistical Analysis

In each experiment, the experimental units correspond to the amended Andisol (SO, S100, S200 and S300) contained in the bags, which are extracted from sub-samples for each analysis, either for chemical characterization, C-C02 evolution or for biological analysis. Then, correlation between the chemical and biological properties was carried out. In the correlation analysis, the global behavior of biological and chemical analyses was considered in each treatment during the evaluation time. These results were discussed by variance analysis and multiple range test of Tukey, with prior Levene test application for variance homogeneity.






Chemical analyses of Andisol under LCM application

The chemical characterization of the amended and non-amended Andisol was determined over 60 days of incubation (Table 1). The incorporation of increasing LCM dose modified the soil chemical properties.

The S300 treatment always showed differences in respect to the other LCM dose. On incubation day 1, the pH values varied significantly. After 20 incubation days, SO, S100 and S200 were similar, while S300 continued to present a significant difference as compared to the other treatments throughout the 60 incubation days. The OM content on day 1 of incubation presented significant differences between SO and the amended soils. After 5 days S100 was similar to SO.

There were no significant differences after 60 days. The contents of N and P from on day 5 of incubation presented no significant differences between SO, S100 and S200. However the N and P content of S300 remained at high levels throughout the whole incubation period. The EC, K content and DOC presented significant differences between the amended soils during the whole incubation period, showing higher values at the highest dose of LCM.

The pH variation of the amended soils may be explained by the LCM pH close neutral. An increase in the pH of S300 is possibly due to higher exchangeable bases content in this treatment (Valarini et al, 2009). Other studies have reported a similar effect on soil pH after application animal manure (Aguilera et al, 1995; Min et al, 2003; Eghball et al, 2005; Salazar et al, 2007). The increase of pH has been attributed to buffering effect and organic acid in cattle manure (Whalen et al, 2000). The EC was higher in the soils with a greater LCM dose due to the high content of soluble salts present in the added amendment in the present study (Plaza, 2004). The increase in OM resulting from the application of LCM is due to the addition of external OM with a high C content and the efficient metabolic activity of the microorganisms present in the soil (Oades, 1984; Bare, 1992; Islam and Weil, 2000; Min, etal, 2003).

Studies carried out in our laboratory indicate that the addition of LCM in increasing dose produces an initial rise in DOC, which falls to its initial condition after 30 days in amended soils S100 and S200, due probably to the stabilization process as C transformation by microorganisms and adsorption process of DOC in soil. However in S300 it remains higher than in the soil to which no LCM was applied. This increase has beenassociated with higher DOC content in S300 (Briceño et al, 2008).

Differences in respiratory activity

The results indicated that the incorporation of LCM has an effect on C-C02 evolution, with a tendency for greater accumulated C-C02 evolution as the LCM dose increases, it was observed that SO < S100 < S200 < S300. For the highest dose of LCM (S300), the maximum value obtained for accumulated C-C02 was 2,092.20 ug C-C02 g-1; for S200, the value was 1,701.44 μg C-C02g-1; for S100, it was 940.88 μg C-C02 μg-1; and for the soil without liquid manure 632.89 μg C-C02 g1. Significant differences occurred between SO, S100, S200 and S300 for all incubation times (Table 2).

The C-C02 evolution rate constant showed that the amended soils presented a higher C-C02 evolution on day 1 of incubation. There were significant differences between the treatments, presenting S300 the highest rate of respiration. The C-C02 evolution rate constant (day-1) of the amended soil decreased to similar levels to those presented by non-amended soils after 10 incubation days (Figure 1).

Studies carried out in agricultural soils which have been planted repeatedly with maize have reported similar behaviours when mineralization was studied. They showed that the highest production of C-C02 was generated showing the highest production of C-C02 being generated in the soils which presented the highest microbial activity (Barriuso and Houot, 1996).

According to the respiration rate the highest level of respiration in this study was obtained on day 1 of incubation, which coincides with studies done by Barriuso and Houot (1996). This occurs because when LCM is applied, anamendment is being added which has easily degradable substrates, various forms of C available, and a large quantity of exogenous microorganisms (Mondini et ah, 1997; Perucci, 1992). All this aspects can increase microbial activity and the biodegradation of the OM in the soil (Plaza, 2004)


Soil enzymatic activities   

The soil enzymatic activities are  summarized in Table 3. The LCM incorporation caused an increased in the  FDA hydrolysis of amended soils.  However, there was less initial activity in  the amended soils than in the non-amended soil during the first 3 incubation days.


The FDA hydrolysis increased from 537.42 (ig fluorescein g-1 h-1 representing the soil initial activity, to 1145.93 μg fluorescein g-1 h-1 and 1100.52 μg fluorescein g-1 h-1, after 20 evaluation days in the S200 y S300 treatments, respectively. These values decreased at the final evaluation; however S300 remained with a higher value.

The behaviour of FDA hydrolysis on days 1 and 3 of incubation may be explained as an enzyme inhibition produced by the addition of the amendment to the microbial biomass, which from day 5 would begin the production of hydrolytic enzymes as a symptom of this effect. Some of these enzymes might be of endocellular origin, derived by cell lysis (Perucci et al, 2000). The increase in the FDA hydrolysis after 3 incubation days indicates a higher activity of the microorganisms due to nutrients contribution, pH increment and OM present in the LCM. These factors contributed to the development soil microorganisms (Gallardo e al, 2010).

Other study relates FDA hydrolysis to the degree of composting of the amendment added (Sánchez-Monedero et al, 2008), and also suggests that it can be used to measure of the hydrolytic activity of the microorganisms in the soil (Dumonet et al, 1993). The fluorescein diacetate hydrolysis becomes stabilizes again when the OM in an amendment becomes stabilized, since a smaller quantity of hydrolytic enzymes is available (Perucci et al, 2000). The stabilization process may be considered as the transformation of C through the mineralization carried out by the microorganisms in the soil (Kalbitz et al, 2003), which lead to a constant activity of the microorganisms.

In the present study, the (β-glucosidase activity showed a significant effect (P≤0.05) of LCM application (Table 3). The (β-glucosidase activity increased from 5.9 (μg p-NP g-1 h-1, representing the activity of the non-amended soil, up to 10.2 μg p-NP g-1h-1 in the S300 treatment, after 20 incubation days. These values decreased over the 30 incubation days, except for S300 treatment.

The (β-glucosidase activity has been evaluated in soils from southern Chile under different tillage system being positively related with the total soil carbon (Alvear et al, 2005). The results obtained in this study showed that (β-glucosidase activity increased as amendment was added to the soil, coinciding with the findings of Deng and Tabatabai (1996), who found that the activity of this enzyme rises with increased accumulation of OM and carbon content in the soil. Various studies indicate that (β-glucosidase activity is related to the soil OM content, its humidity and the transformation produced by the microorganisms to obtain energy. The application of liquid cow manure provides a source of available C which serves as a source of energy especially for enzymes related to the carbon cycle (García-Gil et al, 2004).

Correlation Analyses between chemical and biological amended Andisol properties

The physicochemical properties were analyzed in correlation with the C-C02 evolution and with FDA hydrolysis (Table 4).


A significant inverse correlation was found between the C-C02 evolution in amended soils and their pH values, with S200 and S300 presenting the greatest correlation, indicating that as pH values reduced over time, there was greater C-C02 evolution.

The EC and the C-C02 evolution of the S200 and S300 treatments also showed a significant inverse correlation, indicating that the C-C02 evolution increased as the EC values diminished. A positive correlation was found in the analysis between C-C02 and OM, suggesting that the C-C02 evolution of all the amended soils was greater when the OM content was higher. For the elements N, P and K, there was no correlation between C-C02 and P, however a significant inverse correlation was found between C-C02 and the N content, and a high positive correlation was shown between C-C02 and the K content in all the amended soils.

There was no general trend of correlation with respect to FDA hydrolysis, except between FDA hydrolysis and the P content, for which S200 and S300 showed the highest significant inverse correlation. These relations would indicate that the chemical properties influence microbial activity specially those evaluated by FDA hydrolysis and C-C02 indicators.

Studies carried out by Plaza (2004) and Albarrán et ah, (2004), who used amendments originating from pig production, found that the application of OM added easily degradable compounds, which favours microbial activity and the biodegradation of the OM. Furthermore the incorporation of LCM generates an initial ammonification process as described above and an increase in the concentration of the soluble salts which characterize the LCM (Plaza, 2004).

In the present study a higher respiration rate was found at the start of incubation, which coincides with the greater OM values, subsequently these values start to return to soil values and the respiration rate starts to fall, which would occur due to the stabilization processes occurring in the soil (Kalbitz et al, 2003). According to previous studies, this process starts 10 days after the application of the LCM using the same soil (Aguilera et al, 2009).



The incorporation of increasing dose of LCM modified soil chemical properties. High dose of LCM application resulted in increased the pH values, EC, OM content, nutrients and DOC. The highest amendment dose presented a significant difference as compared to the other treatments throughout the 60 incubation days. The respiration activity of the amended soils increased with the increasing dose of LCM. The C-C02 evolution rate constant showed that amended soils presented a higher C-C02 evolution levels on 1 incubation day. The amended soils which were incubated for more than 10 days presented a constant rate of respiration. The soil enzymatic activities indicated that LCM incorporation caused an increased in the FDA hydrolysis and the β-glucosidase of amended soils. Although there was an initial inhibition in the FDA hydrolysis.

In the study it was found that the incorporation of increasing dose of LCM modified the chemical and biological properties of the soil and that there is a correlation between the chemical properties of the soils amended and the biological activity, mainly C-C02 evolution. An adequate LCM utilization could to contribute to improve the Management Practices in the intensive dairy systems in southern Chile.



This study was financed by the project FONDECYT 1070568.



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