Crude glycerol co-digestion associated with swine manure in biogas production: A study in Brazil

1 Unioeste – Rua Universitária 1619, Jardim Universitário Cascavel – PR, Brazil. ZIP Code: 85814-110, Brazil. 2 FAG Avenida das Torres 500, Loteamento FAG – Cascavel – PR, Brazil. ZIP Code: 85804125, Brazil. 3 Unioeste Rua da Faculdade 645, Jardim La Salle – Toledo – PR, Brazil. ZIP Code: 85903-000, Brazil. 4 Lene – Laboratório de Energia Rua Universitária 1619, Jd. Universitário Cascavel – PR, Brazil Unioeste, Brazil. 5 UFPR, Rua Pioneiro 2153 Jardim Dallas – Palotina – PR, Brazil. ZIP Code: 85950-000. 6 Unioeste Rua Pernambuco1777, Centro –ZIP Code: 858960-000, Brazil.


INTRODUCTION
Environmental concerns about climate change in recent years have gained international significance, taking place on the development agenda.Being cited as the main factor responsible for this fear with the environment, emissions of greenhouse gases have been the target of environmental policies of different countries (Nyko et al., 2010).
The political and economic crisis in oil-producing countries has led importing countries to seek investment seriously in research and development of alternative solutions to reduce its dependence on this ore.One alternative has been the use of vegetable oil, which are bio-fuels, instead of diesel oil.In seeking the reduction of dependency of petroleum and reducing environmental impacts, there were alternatives like biodiesel, leveraged by the National Program and Production and Use of Biodiesel (PROBIODIESEL).The law no.11,097, of January 13, 2005, introduced the biodiesel in the Brazilian energy matrix (Brazil, 2005).However in the production of biodiesel through any trigly ceride is a by product generated approximately 10 kg of glycerol generated for each100L of produced biodiesel (Santibanez et al., 2011).Due to its composition rich in carbon and easy degradation, glycerin has properties conducive to an aerobicco-digestão being susceptible toits association with swine manure, which is are sidue of the digestion wide spread technology.The growth of pig production in southern Brazil, has made the country alcançace the third in the world (ABIPECS, 2012).The intensification of pig farm because of its large geographic concentration, leads to a high concentration of manure, even with its high content of nutrients, it can become a dangerous pollutant both terrestrial as aquatic (Segat et al., 2015).The intense deposition in the soil of swine manure can cause leaching of the following nutrients P, K, Cu and Zn (Scherer et al., 2010).
The practice of co-digestion has benefits as increased load easily degradable organic matter, dilution of toxic substances, improving the mixing buffer capacity, increased biogas production, a better quality of a digested product, and reduced cost, better balance nutrients and a possible synergistic effect of microbial consortia on the efficiency and yield offer mentation (Alvarez et al., 2010;Ashekuzzaman and Poulsen, 2011;Rao and Baral, 2011;Khalid et al., 2011;Esposito et al., 2012;Wang et al., 2012;Zheng et al., 2015;Pagés-díaz et al., 2015;Khoufi et al., 2015).The Program and Biodiesel Production and National use determined the addition of biodiesel to diesel in incremental scales in 2010 reached 5% in 2015 and rose to 7% (Brazil, 2015;ANP, 2015).This increase results in a large increase in the supply of a co-product generated in the production of biodiesel, which is glycerin.This will generate a surplus that the current market cannot absorb, reflecting the need for alternatives to the use of this coproduct in a sustainable way, both economically and environmentally (Siqueira, 2012).
Given the earlier explanation and in order to avoid future problems arising from the accumulation of glycerol and to make it more competitive, biodiesel production justifies the search for the utilization of alternative crude glycerol generated in this production.Therefore, this study aims to evaluate the efficiency of crude glycerol, from the production of biodiesel, in obtaining biogascodigestion pig manure.Thus, it becomes necessary to improve the different operating parameters involved in the anaerobic fermentation process for obtaining biogas from the crude glycerol producing clean, renewable energy and contributing to the Brazilian energy matrix.

MATERIALS AND METHODS
The experiment was conducted in the Development Center and Technology Dissemination (CEDETEC) Faculty of Assis Gurgacz (FAG) in the city of Cascavel Paraná State, Brazil.The crude glycerol was courtesy of a biodiesel plant, also the city of Toledo, its origin comes from soybean oil of methyl trans esterification.
The swine manure were provided by Granja Scheid, located in Sanga Line Guarani, in the Good Principle district of the city of Toledo -PR.The property has about 600 pigs and produces an average of 3 m 3 / day of waste.Pigs are raised in pens and these facilities are housed in collective cages, with about 30 animals each.The pen is made of brick and was built in such a way as to facilitate the management of waste, with concrete floors sunken directed to a collection box, avoiding thus contamination of neighboring areas.The waste was collected and preserved in fresh thermal polystyrene box (STYROFOAM) until they are needed.
In order to analyze the samples, micro-reactors made with glass containers with a volume of50mLand equipped with airtight 10mLsyringe, whose function was to measure the volume of produced biogas (Azevedo, 2010) were used.In micro-reactors added to the swine manure in concentrations of 20, 40and 60%, the culture medium comprisingly cerol in concentrations of 0, 5, 10, 20, 30 and 40% w/w and minerals solution (nutrient), as can be seen in Figure 1a.The micro-reactors were placed in 21 days-culture green house, as can be seen in Figure1b.The biogas volume was assessed daily by observing the displacement of the syringe plunger.
The culture medium was composed of crude glycerol (C3H8O3) in concentrations of 5, 10, 20, 30 and 40% w/was a carbon source and mineral salts solution (nutrients).The solution of mineral salts followed Azevedo methodology(2010), where in 1 L of distilled water diluted to 2 gdi basic potassium phosphate (K2HPO4), 20 g monobasic potassium phosphate(KH2PO4) and 3.5 gurea ((NH2) 2CO).The experiment was conducted without variation in pH.For the pH no variation in the samples was added3 mlKOH10Ninthe original culture medium yielding pH 7.2.The experiments were placed in the culture oven at 35°C for21 days.Total solids (TSs), refers to the remaining waste material into the container after evaporation and drying of the sample.The analysis of total solids (TSs) was determined according to the methodology described in American Public Health Association-APHA (1992).
First were measured capsules porcelain leaving them in oven at (55±50°C) for 1 h, followed by cooling in desiccator and weighing to the nearest 0.1 mg.Then transferred to10 ml of capsules pig manure, the culture medium comprisingly cerol (at concentrations of 0, 5, 10, 20, 30 and 40% w/w) and the solution mineral salts (nutrients) being subsequently placed in a greenhouse at a *Corresponding author.E-mail: ksanderson@fag.edu.brAuthor(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License  temperature of 103 to 105°C for about 24 huntil constant weight.Withdrawal greenhouse was placed in a desiccator to cool and the capsules were re-weighed, thus obtaining the final weight (dry) (mp).After 21 days at 35°C was used the methodology described for monitoring the remaining waste material in the micro-reactors.The total solids content was determined by Equation 1: Equation 1: Determination of total solids, wherein: TS=Total Solids; Iw=Initial weight (wet) (g); Fw = Final weight (dry) (g).
After evaluating the performance of micro-reactors was producing a digestion system from laboratory scale to assess the best result.As a reactor used a plastic jug of mineral water to volume of 20L, the mouth was sealed with a rubber stopper with a hole, into which was inserted a hard iron pipe connected to a transparent tube polyurethane for biogas outlet (Figure 2).
After passing through the gas meter, the biogas was stored in a gas sampler (bag).The biogas obtained was evaluated in relation to the produced volume.Methane proportions were analyzed by agas chromatograph.After 30 days, the same procedure described earlier was used, but there actor was fed with only manure and mineral salts solution.

RESULTS AND DISCUSSION
Figures 3, 4 and 5 show the behavior substrate at concentrations of 0, 5 (w/w), 10 (w/w), 20 (w/w), 30% (w/w) and 40% (w/w) crude glycerol in concentrations of  20, 40 and 60% swine manure on the accumulated biogas production.Looking at Figures 3, 4 and 5, it is seen that the sample With 5% glycerol when added in concentrations of 20, 40 and 60% swine manure significantly increased the volume of produced biogas, reaching the twentieth first day with 16.17 ml to 20%, 27.13 ml of 40% and 52.52 ml for 60% of swine manure.
Samples with 10% glycerol concentration biogas obtained a slightly higher, reaching a volume of 10.33 ml  The process of the daily biogas production in these concentrations was slower, indicating an inhibitory substrate trend.Azevedo (2010) in his studies on the effect of the concentration of glycerol for the production of biog as also found that the concentration of 40% (w/w) was inhibitory in all experiments.Fountoulakis et al. (2010) comment on their work that inhibitory trend in the biogas at high concentrations of glycerol can be associated with contamination of glycerol for biodiesel residues or an organic over loading the digester.The results show that at this stage anaerobic digestion with the addition of 5% glycerin and 60% swine manure becomes viable for production of biogas.Backes (2011) in his experiment added 6% crude glycerol in swine manure and noted a significant increase in biogas production, obtaining average production of biogas at about 0.0121m 3 and was significantly higher when compared to control (± 0.0035m 3 ) and treatment with 3% glycerol (± 0.0065m 3 ).Analyzing the data in Table 1 (21 days of treatment), it is observed that the percentages add glycerol in concentrations of 5, 10, 20, 30 and 40% per swine manure removal rate total solids (TSs) declined over the treatments, with the greatest reduction in the control treatment, without glycerol(0%), reaching 30.40%.Since treatment with 5% glycerol reduction was 28.70%.Thus the treatment without glycerol (0%) and 5% glycerin, after 21 days at 35°C.They showed almost the same performance in reducing the mass of solids.
The results presented here show that the topical use of5% glycerol in anaerobic digestion of swine manure is effective in removing total solids (TSs).Backes (2011) in his study of anaerobic digestion noted that reductions in total solids of cattle manure after the procedure decreased to almost all treatments(control, 6 and 9%), with the largest reduction occurred in the control treatment, reaching 28%, and treatment with 9% glycerin to15%.Konrad (2010) in relation to the generation of biogas, obtained in the control sample (0% glycerol) a production of biogas 0.0024m 3 , while the sample with 3% glycerin, production amounted to about 0.01292m 3 of biogas, which it represents an increase of 81.4% over the control sample.Since the biogas production with the addition of6% glycerin resulted in a total yield of 0.0058 m 3 , representing 59.2% increase relative to the control sample.
From the results, the study came to the conclusion that the anaerobic digestion with the addition of 5% crude glycerin and 60% of swine manure is feasible for biogas production.Thus, it played up a laboratory reactor, and it was possible in order to analyze the volume of produced biogas and compares it with the control treatment.The test results are shown in Figure 6.Treatment with addition of 5% crude glycerin was increase in biogas production compared to the control treatment.The total gas production in this treatment was 0,373m 3 of biogas while in the control treatment (0% glycerin), was 0,127m 3 of biogas.Figure 7 illustrates the chromatogram of the samples containing 0 and 5% (w / w) of glycerin.
The chromatogram shows peaks corresponding to air, methane and carbon dioxide, respectively.
The retention times of methane and carbon dioxide of samples showed similar behavior to the standards.Since the peak of the air samples as compared to standard, they showed lower values being positive for the process.The air is lethal to anaerobic bacteria.If oxygen in the atmosphere, anaerobic bacteria paralyze their metabolism and cease to grow, they are responsible for the production of methane.By chromatographic analysis (Figure 7) it was possible to quantify the existing methane content in the samples studied.The results of this experiment showed that the sample methane percentage with 5% glycerin and control samples (0% glycerol) with 60% swine manure have similar values, and the sample with 5% glycerol obtained a value stronger than the control sample, reaching a plateau with content of 58.87% methane, while the control sample has reached 58.45%, as shown in Figure 8.
According to Souza et al. (2007), the biogas composition varies depending on the material from which it originated, but its basic composition is 40 to 70% methane (CH 4 ), 30-40% carbon dioxide (CO 2 ), nitrogen traces, hydrogen and gas hydrogen sulfide.Oliveira and Higarashi (2006) reported values between50 to 70% methane.Thus, the values found in this study are consistent with the values found in the literature.Konrad (2010) in their experiments with sludge treatment plant to quantify the biogas in the control sample (0% glycerol) had levels of methane of 68%.In the sample in which 3% glycerol added methane values reached 72% being  satisfactory for energy purposes.
Since methane percentage with the addition of6% glycerol was 61%.Backes (2011) adding 3% glycerol swine manure obtained an average of 68% methane.In the control sample, 60% methane content was obtained.In the sample with 6%, glycerin value was around 90% methane.In his studies Chen et al. (2008) added glycerol to the manure and there was increased production of biogas and methane.The average content of methane in the biogas was 63to 70%.

Conclusion
The results showed that the amount of swine manure has great influence on the production of biogas.In this study, the concentration with higher yields in biogas production was the sample with60% of swine manure.The crude glycerol associated with the manure in a ratio of 5% w/w can be used as a supplement in anaerobic digestion, providing a significant increase in the production of biogas.
By increasing the concentration of glycerol were observed difficulties and little production of biogas.The process was slower, indicating an inhibitory substrate trend.It follows that the amount of glycerol added to the anaerobic digestion should have a limiting level of concentration and time to adapt to the biocenose, by avoiding an organic overhead in the process.The addition of 5% glycerol in the anaerobic biological treatment system with pig manure was efficient in reducing total solids, the read aptation of microorganisms involved in anaerobic digestion process with the substrate.The biogas composition was not altered by the presence of 5% glycerol methane which aspect ratio remained virtually unchanged compared with the control sample.

Figure 1 .
Figure 1.Micro-reactorsused in their production and micro-reactors conditioned in the greenhouse (a.Micro-reactors used in the production packaged of biogas; b.Greenhouse with micro-reactors).

Table 1 .
Total solids (TSs) and their respective reduction ratios after 21 days.