Study of stillage biodegradation by respirometry in sandy and clay soils

1 Department of Biochemistry and Microbiology – Institute of Biosciences São Paulo State University, UNESP, Av. 24A, no 1515, Bela Vista, CEP 13506-900, Rio Claro, São Paulo, Brazil. 2 Institute of Geosciences and Exact SciencesSão Paulo State University, UNESP, Brazil. 3 Department of Statistics, Applied Mathematics and Computation Institute of Geosciences and Exact Sciences-São Paulo State University, UNESP, Brazil. 4 Research Center Mokiti Okada (CPMO)São Paulo – Brazil.


INTRODUCTION
With rising concerns about keeping natural environments in good quality, all effluent or composite able to pollute or contaminate it must be treated efficiently.The alcohol production in Brazil is continuously extended according to the statistical data by UNICA (2009).Between the crops of 1997/1998 and 2007/2008, there was an increase of 62% in production, corresponding to a raise of 190 million tons of sugarcane produced in the country.Studies shows that each ton sugarcane can produce, on average, 80 L of ethanol (EtOH) and each liter of EtOH produced generates, on average, 13 L of stillage (Nicochelli et al., 2012).In Brazil, 21.1 billion liters of ethanol were produced in 2012 (Renewable Fuels Association, 2012).According to MAPA (2012), there is a positive projection for the next years of 58.8 billion liters in 2019, more than double of what was produced in 2008.
This fact is mainly connected to the increase in the number of vehicles powered by biofuels (vehicles that can be fueled both with ethanol and gasoline), besides the intern and extern consume of anhydrous alcohol.As stillage is the waste of EtOH distilleries, when it is discarded in the environment incorrectly, it can constitute a serious polluting source.However, this sub-product of distilleries can be well used as a fertilizer substitute in agriculture, as a complement for animal feed or in the production of biogas.
According to Lelis Neto (2008), stillage is characterized as an effluent with a high polluting power, about a hundred times more than domestic sewage.This is due to its organic matter richness, low pH, high capacity for corrosion and high indexes of chemical and biochemical oxygen demand (COD and BOD, respectively).The high temperature in the exit distillers compromises the immediate application in the soil.Because of its composition, it is considered harmful to the fauna, flora, freshwater micro-fauna, besides chasing away the marine fauna which breeds in the mangrove.
The stillage organic matter consists of organic acids, potassium ions and, in less quantity calcium and magnesium.Its nutritional value is related to the origin of the fermentation of the raw material (wort).When the wort is from molasses (residue of the sugarcane production), the stillage presents higher concentration of nutrients, which falls when the wort origin is from sugarcane juice (Rossetto, 1987;Kannan and Upreti, 2008;Acharya et al., 2008).
With the intention of lightening the sugarcane polluting load, some bioremediation techniques can be used.Those techniques consist in taking care of the contaminated sites with the use of biological agents, such as:-Intrinsic or natural bioremediation: The microorganisms used are autochthonous, that is, from the site of interest, without any interference of active remediation technologies.-Biostimulation:When there is the addition of stimulating agents, such as nutrients, oxygen and biosurfactants.-Bioaugmentation: Consists in the inoculation of enriched microbial consortia (Bento et al., 2003).
The bioremediation corresponds to physical, chemical and biological processes for the decontamination of soils and ground water.In order to adopt these processes with safety, it is necessary to develop studies that confirm the pollution reduction in the environment by the limitation of the pollutant displacement aiming the natural attenuation (Chapelle, 1994).
The simulation of field conditions in lab experiments allows to verify the efficiency of the bioremediation process by producing relevant results for enviromental risk management.Respirometry is a technique for assessing the biodegradation process, based on an aerobic system where the quantity of CO 2 produced and oxygen consumed shows how easy it is for microorganisms to degrade the organic matter present in the residue and, then, the potential use of the bioremediation process for the recovery of contaminated areas (Costa et al., 2009).
Thus, the major benefit of this process is the evaluation of the pollutant mineralization, that is, how it transforms into carbonic gas, water and biomass.In these tests, it is possible to include the total count of heterotrophic microorganisms or specific substrate degraders and also the degrading rates corresponding to the pollutant disappearance to confirm the degrading process (Kataoka, 2001).
The Respirometric method by Bartha and Pramer was used in this study, applied in works that studied bioremediation in contaminated soils and recommended by NBR 14283 (ABNT, 1999) to determine the biodegradation of residues in soils by the resperometric method.The aim of this work was to evaluate and quantify the stillage biodegradation in sandy and clay soils, besides verifying the efficiency of the inolucum embiotic Line as an accelerator of the biodegradation.

Soil characterization and sampling
The samples of soils were collected in February 2012 according to the technical rule L.6.245 from Cetesb (1984), in the geographic coordinates: (23 k 240122.10mE 7524445,61m S) and (23 K 238189,08m E 7520899,96m S) localized in the municipality of Rio Claro, in the State of São Paulo, Brazil.These samples were taken from the soil superficial layer of non-contaminated places.Table 1 shows the physic-chemical characteristics of both soils.

Bartha and Pramer respirometric method
Biodegradation experiments, according to the Bartha and Pramer Respirometric Method (1965), were carried out in Bartha biometer flasks (250 ml) used to measure the microbial production of CO2 (Table 2).For each type of soil, flasks were prepared in triplicates with 50 g of soil and addition of stillage or water with and without inoculum according to the protocol (Table 3).Samples were incubated at 27°C in the dark.The quantity of stillage added to the treatments was of 16% and they were properly adjusted for 60% of water retention capacity for each soil.
Quantification of CO2 production generated in the respirometric experiment and the calculations of biodegradation efficiency obeyed the technical rule L6350 (Cetesb, 1990).

Inolucum (embiotic line) microbial count in colony forming units (CFU/ml)
After the inoculum activation, the bacteria count was carried out by "Pour-Plate" technique in PCA environment with addition of actidione (5 ppm), according to the technical rule L. 5.201 (Cetesb, 1986).In order to count the fungi and yeasts, "Spread-Plate" plating was carried out in Sabouraud Dextrose Agar environment (SDA) with addition of antibiotic (ampicillin and nalidixic acid, 5 ppm).The plates with bacteria were kept in greenhouse at 35°C and the ones with fungi and yeasts at 28°C.

Stillage microbial count
The sample was progressively diluted and plating was carried out by obeying the same procedures from inolucum (embiotic line) microbial count in Colony Forming Units (CFU/ml).

The soil initial and final microbial count
Samples of 10 g of each soil were placed in 90 mL of saline solution (0.85%), and flasks were shaken for 20 min.Afterwards, serial dilutions and platings were carried out according to inolucum (embiotic line) microbial count in Colony Forming Units (CFU/mL).The microorganisms were calculated by counting of colonies in UFC/g of dry soil.

Statistical analysis
Friedman test (Zar, 1999;Ayres et al., 2007) was applied in two ways.For the inoculum performance verification during the biodegradation process and for the verification of the differences observed in this process for both soils involved in the experiment.

Bartha and Pramer respirometry
Figures 1 and 2 indicate the increase of CO 2 production in the treatments where stillage was added, showing the pollutant biodegradation activity.In the beginning of the process, in sandy soils, there was a discreet increase of CO 2 production in the treatment inoculated with embiotic line, which was evidenced in the peak of CO 2 production.However, this effect was not maintained.Through the development of the experiment, CO 2 rates decreased when compared to the treatment without embiotic line (Figure 1).In clay soils, the CO 2 production curve for the treatments with and without the product remained very close (Figure 2).According to Rafaldini et al. (2006), who tested effective microorganisms (EM), from the same manufacturer, in stillage reservoir tanks there was not considerable biodegradation acceleration either.
Analyzing the beginning of the biodegradation process,   Figure 3 records the daily production of CO 2 , indicating that there was a considerable difference in the biodegradation process evolution between the sandy and clay soils.It was observed that in the clay soil the biodegradation began immediately and achieved superior levels of CO 2 production when compared to the sandy soil.Certainly, this is due to the greater capacity of liquid and nutrient retention and the presence of autochthonous microorganisms in clay soils.According to Lelis Neto (2008), potassium, nitrate and calcium ions show higher interaction with the solid fraction of that soil, when compared with sandy soil, being retained more easily in the clay soil.The fast stillage biodegradation, just in the first days of the experiment, could inhibit the organic matter leaching, avoiding the contamination of deeper soil layers.According to Lelis Neto (2008), with reference to nitrate percolation, neither soils present risks of groundwater contamination, ever for concentrations of 300 m 3 /ha.By analyzing the cumulated production of CO 2 (Figure 4), it was observed that the clay soil gotten both in the treatment with stillage and in the control with water, a CO 2 level superior to the treatments of sandy soils, evidencing the real difference in the biodegradation process between both soils.

Biodegradation efficiency
On the second day of the trial, the biodegradation efficiency the clay soil achieved more than 30%.However, in the sandy soil this index is achieved from the third day on.Although there are significant differences related to the biodegradation of the stillage organic matter in both soils studied, as the statistical tests evidenced, the efficiency in the mineralization of this pollutant achieved 92% in clay soils and 89% in sandy ones in 50 days of trial (Figure 4 and Table 4).Both results were considered excellent with reference to the effluent biodegradation, once the law determines a minimum of 30% efficiency of biodegradation for residue disposal in the soil (NBR 14283-ABNT, 1999).

Initial microbial count
When analyzing Table 5, which registers a microbial count, it was verified that both soils had yeasts and fungi on the order of 10 4 UFC/g and bacteria on the order of 10 5 UFC/g.The clay soil presented a slightly superior number of microorganisms.

Final microbial count
By comparing initial and final UFC/g quantities of soils in Table 6, we can verify that the quantity of microorganisms was not greatly modified in treatments with water and with water added of inoculum.In the beginning of the process there was an increase in the  metabolic activity of the microorganisms, and then this activity started decreasing due to the environment depletion.At the end of the trial, the number of microorganisms was smaller or close to the initial one.
In the treatments with stillage, the beginning of the process showed an intensive metabolic activity.The stillage used as a source of organic matter enriched the soil, stimulated both the increase of metabolic activity and the increase and maintenance of the microorganisms population.Therefore, the number of bacteria was higher at the end of 50 days.This increase in the bacterial population was also verified by Mariano et al. (2009).Clay soil presents a larger amount of organic matter, water retention capacity and a great number of microorganisms because of its colloidal characteristic, due to the presence of clay.Therefore, it converted both soil and stillage organic matter until its mineralization more efficiently.With reference to the sandy soil, as it has a lower water retention capacity, it presents less organic matter, a smaller number of microorganisms, therefore stillage biodegradation in this kind of soil became less efficient.

Friedman test
When the treatment data with and without the embiotic line are compared, statistical tests showed that in sandy soils there was a significant decrease in the stillage biodegradation when the inoculum was used (p=0.036).
In clay soils the use of the product didn't vary the biodegradation significantly (p=0.1456).However, the comparison made between the biodegradation process performance in sandy and clay soils demonstrated significant differences in both the control treatment, only with water (p=<0.0001),and with treatments with stillage (p=0.0153).Thus, this proves the major biodegradation capacity of the clay soil when comparing to sandy soil.

Conclusion
With this study it was verified that both soils present good levels of stillage biodegradation.However, clay soils, which present greater field capacity, have higher water, organic matter and microorganism retention capacity, provided a higher efficiency in biodegradation of this effluent, verified statistically.Regarding the use of the embiotic line, the experiment showed that this product doesn't interfere positively in the stillage biodegradation for either soils, with possible adjustments to its composition being necessary.

Figure 1 .
Figure 1.Daily production of CO2 in sandy soil during 50 days of incubation according to the respirometric experiment.

Figure 2 .
Figure 2. Daily production of CO2 in clay soil during 50 days of incubation according to the respirometric experiment.

Figure 3 .
Figure 3.Comparison of the daily CO2 production in sandy and clay soil during 20 days of incubation according to the respirometric experiment.

Figure 4 .
Figure 4. Comparison of cumulative CO2 production in sandy and clay soil during 50 days of incubation according to the respirometric experiment.

Table 3 .
Respirometric experiment for sandy and clay soils.

Table 4 .
Biodegradation efficiency (%) of stillage for 50 days in clay and sandy soils.

Table 5 .
Initial microbial count.Results for the inoculum (embiotic Line) and stillage are expressed in CFU/ml (Colony Forming Units per ml) and those for sandy and clay soils in CFU/g (Colony Forming Units per g).

Table 6 .
Final microbial count for sandy and clay soils in CFU/g (Colony Forming Units per mL gram of soil).