ORIGINAL ARTICLE

Study of Methane Potential in Three Bovine Pastures Pre-Treated with Enzymes

Dr.C. Carlos M. Martínez Hernández^{I  *} 

Dr. Hans Oechsner^II 

Dra. Annett Reinhard^II 

Dr. Yaser García López^I 

MSc. Arnaldo Martínez Hernández^I 

^I Universidad Central “Marta Abreu” de las Villas, Santa Clara, Villa Clara, Cuba.

^II Universidad de Hohenheim, Landensanstalt fur Agrartechnik und Bioenergie, Stuttgart, Germany.

ABSTRACT

In this work, it was studied the influence of ZY maXX XL 200 enzyme pre-treatment of three agricultural substrata (mulatto pasture, natural pasture and variety CT-115), on their potentiality for methane production. The pre-treatment consisted on the addition of an enzyme (ZY maXX XL 200) in dose of 100 µl/syringes for 350 mg of substrate in 30^-1 mL of bovine inoculum. The results of the substrates bio digested were compared with the ones obtained without the addition of the investigated enzyme. In the three cases, significant increments of specific methane yield were achieved. The best result was obtained with mulatto pasture (0.268 m³ CH₄/kg VS), followed by the variety CT-115 (0.258 m³ CH₄/kg VS) and culminating with natural pasture (0.254 m³ CH₄/kg VS). Nevertheless, increments were observed when the pre-treatment was used with enzymes and bovine inoculum vs. when it was not used. On the other hand, the pH had a similar behavior in all substrates where the pre-treatment was applied, as well as where it was not used.

Keywords: Biomass; agricultural wastes; enzyme pre-treatment

INTRODUCTION

The composition of the substrates is the main factor to determine the yield and methane potential. Most of the bibliographical sources report that the differences in the kinetics, potential and methane yield depend on the type of substrates utilized (^{Forster et al., 2012}). The methods of substrates pretreatment have as object, to improve their anaerobic digestion qualities by altering their physical, chemical and biological properties. In spite of that, they show certain particularities such as increasing of the manipulation costs, increment of legislative requirements for stabilization and removal of possible pathogens, tendency to handle smaller nitrogen limits, which allows handling the age of these substrates and increasing the biodegradability of activated substrates (^{Zhong et al., 2011}). Consequently, it is necessary to analyze the pre - treatments to use depending on the type, performance and costs.

Pre-biological treatments: the objective of the pre-biological treatments are to prepare the substrates for the enzymatic degradation and the best method and pre-treatment condition depends significantly on the substrates type. Among the utilized microorganisms to degrade organic substrates there are several types of mushrooms, such as: Carmelite, white and soft rotten, besides some types of bacteria’s (^{Kurakake et al., 2007}). ^{Taherzadeh and Karimi (2008)} studied biological treatments in office paper with two chains of bacteria (Sphingomonas paucimobiles and Bacillus circulans), obtaining improvements in the enzymatic hydrolysis, as well as 94% of recovery of sugar. Depending on the substrate type (residuals of houses, waters of industries, residuals of still etc), the enzymatic attack can be carried out by different types of mushrooms or combinations of these (Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Aspergillus terreus, etc).

^{Taniguchi et al. (2005)}, evaluated pre-biological treatments in rice straw using 4 white-rotten mushrooms (Phanerochaete chrysospurium, Tramete versicolor, Ceriporiopsis subvermispora and Pleurutus ostreatus) and the pre-treatment with Pleurutus ostreatus generated a selective degradation of lignin and an increment of the susceptibility of rice straw to the enzymatic hydrolysis. In addition, in the phase of solid fermentation of orange shell with chains of mushrooms, type Sporotrichum, Aspergillus, Fusarium and Penicillum, the constituent feeding capacity was improved and the level of antimicrobial substances was reduced. In a similar work ^{Taherzadeh and Karimi (2008)}, used white-soft mushroom cultivations to decontaminate waste waters of olives milling, improving their digestion. ^{Vintiloiu et al. (2009)}, investigated the influence of temperature and pH of several commercial enzymes on the degradation of corn ears and straw. According to these authors, the best effects were obtained at 50 ⁰C and the enzymes originated by mushrooms present their best potentialities to pH values between 4 and 6. However, the methane genesis takes place to values between 6 and 8, therefore, it would be necessary to look for an enzyme that maintains a good activity in these pH ranges.

Low energy requirement, no use of chemical and gentle environmental condition are the main advantages of the biological pre-treatments. However, the efficiency of these pre-treatments is sometimes low. For such a reason, the pre-biological treatments need of an extra state that assures the later enzymatic attack to these; this alternative can be carried out through previous pre-treatments such as physical or chemical.

^{Martínez et al. (2015)} used a chemical-thermal pre treatment in some of the biomass that are valued in this investigation. Due to that, the present work constitutes a continuation of the use of special pre-treatments to use in the biodigestion of agricultural and of tavern residuals, which have been supported in investigations by ^{Martinez et al. (2014)}, ^{Martinez et al. (2015)} and ^{Martínez and García (2016)}.

The analysis of these bibliographical sources allows appreciating that the application of a pre-treatment with enzymes to organic residuals of type lignin-celluloses is very appropriate to favor the biodegradability and the methane production. This investigation is intended to apply a pre-treatment with enzymes to different types of biomass, studying their effect on biogas production.

METHODS

This work was carried out in the Central University "Marta Abreu" of Las Villas, but the Company BIOPRACT GmbH, through investigators of the University of Hohenheim, Germany, donated the investigation material (Liquid cellulase enzymatic preparation, ZY maXX XL 200). The experimental results, fruit of combined investigation projects, were obtained from September 2017 until November 2018. Different effective norms for this investigation type were consulted as it is indicated next.

The substrates were characterized according to the norm ^{VDI (2006)}and following the characterization and general classification of substrates, the possibilities of ferment ability of the organic materials were determined. The agricultural residuals studied in the Cuban investigation project were sorghum (Sorghum R-132), sorghum (Sorghum halepense), sunflower (Helianthus annuus L JE-94), corn (Zea mays L), malanga (Colocasia esculenta L Schott), sweet potato (Ipomoea sweet potato) and potato (Solanum tuberosum Sw) (^{Martinez et al., 2014}). In addition, a tavern residual was studied (white bread) (^{Martinez et al., 2016}).

Later on, other agricultural residuals were study object such as natural pasture, mulatto pasture and CT-115. In the case of these agricultural residuals, samples were collected taking roots, shafts, leaves and fruits. These residuals were dried off and fractioned in particles of 1 mm of size, taking samples to carry out the investigations on laboratory scale in Germany and on field scale in Cuba. The investigations in Germany were executed in investigation stay developed in the University of Hohenheim between the months of September and November 2017.

In the Germany case, treatment was applied with enzymes and it was investigated with bovine inoculum. In Cuba, these investigations were replied on field scale. A pre-treatment was used with enzymes, the enzyme ZY ma XX XL 200 was added in dose of 100 µl/syringe for 350 mg substrate to value. The substrates inoculated with the enzyme were allowed resting during 4 hours, later on the inoculum was added (30 mL of porcine effluent of an anaerobium biodigester). Next, the substrates pre-treated and the inoculum were introduced in experimental syringes of 100 mL of capacity.

In Germany, bovine inoculum was used (cow manure), in Cuba pig inoculum was used (pig manure) coming from biodigesters in production. In both cases, the experimental syringes, were placed in a denominated addition Hohenheim Bench Test (HBT), to reason of three samples per substrate valued; as well as a sample in plastic container (plastic bottle), in order to investigating the evolution of the pH in the process of anaerobic digestion under field conditions. The object of valuation were the following parameters:

• Dry matter and humidity content according to the norm ^{NC 74-22:85 (1985)};

• Ashy content according to the norm ^{NC 74-30: 85 (1985)};

• Determination of carbon/nitrogen ratio;

• Evolution of the pH in the biodigestion;

• Evaluation of the specific biogas yield.

Starting from the input data, the following parameters were calculated by means of softwares elaborated for these ends (Gärtest nach VDI 4630) and following the norm ^{VDI-4630 (2006)}:

• Biodegradability rate;

• Maximum biogas potential (L/kg SV).

Table 1 shows the test conditions in each biomass under research.

TABLE 1 Test conditions for biomass evaluated prior to the biodigestion process in experimental syringes 

Substrates	Enzyme used	Dose	Hydraulic retention time (HRT= minutes)
Mulatto pasture	ZY maXX XL 200	100 µl/syringes per each 350 mg of substrates in 30^-1 mL Inoculum	120
Natural pasture	ZY maXX XL 200	100 µl/syringes per each 350 mg of substrates in 30^-1 mL Inoculum	120
CT-115	ZY maXX XL 200	100 µl/syringes per each 350 mg of substrates in 30^-1 mL Inoculum	120
Inoculum (cow and pig manure)	Without enzyme pre-treatment

Statistical Analysis

Statistical analysis was performed using the statistical package STATISTICA, STASOFT, 8.0. An ANOVA, One-way analysis and a Tukey HSD test were performed on the specific methane performance variable.

RESULTS AND DISCUSSION

In Figure 1, the evolution of all substrates valued with the addition of enzymes and without them are observed. The best behavior took place with the grass mulatto substrate (0.268 m³ CH₄/kg oDM), followed by the CT-115 (0.258 m³ CH₄/kg oDM) and culminating with the natural grass (0.254 m³ CH₄/kg oDM). Superior increments were observed in all substrates that received the pre-treatment with enzymes with respect to those without the application of the referred pre-treatment. On the other hand, the obtained values are below the ones gotten by ^{Martinez et al. (2014)}. It could be due to the potential of methane of these biomass and to the use of mixtures of roots, leaves, shafts and fruits of these agricultural biomass, which possess bigger quantity of fiber (cellulose and hemi cellulose),compared to the previous studies with these biomass in solitary.

FIGURE 1 Specific methane yield of the biomass studied using cow inoculum and enzyme pre-treatment on laboratory scale in Germany. 

Table 2 shows the results obtained with the substrates evaluated with and without the enzyme treatment.

TABLE 2 Specific yield of biogas and methane. Biomass evaluated with and without enzyme pre-treatment. The results are shown as average ± standard deviation 

Substrate	Biogas content [mL]	Methane content [mL]	Methane content (% of volume)	Specific Biogas yield (m^3/kg VS)	Methane yield specific (m^3 CH4/kg VS)
Bovine inoculum	29±4,3	20±2,8	70±1,0	0,018±0,003	0,013±0,002
Standard hay (Reference substrate)	86±3,0	49±1,5	56±0,5	0,482±0,016	0,273±0,009
CT-115 pasture, without enzymes	114±3,6	64±1,0	56±1,5	0,416±0,014	0,234±0,004
CT-115 pasture, with enzymes	126±7,0	71±4,7	56±1,0	0,461±0,026	0,258±0,018
Mulatto pasture, without enzymes	103±12,5	58±6,5	56±1,0	0,375±0,046	0,211±0,023
Mulatto pasture, with enzymes	134±5,8	74±2,5	55±1,1	0,485±0,020	0,268±0,011
Natural pasture, without enzymes	105±5,2	60±2,0	57±0,5	0,379±0,018	0,217±0,008
Natural pasture, with enzymes	125±11,6	71±6,6	56±0,0	0,452±0,041	0,254±0,023

In Table 2, it is observed that, in CT-115 pasture (the whole plant), mulatto pasture (only leaves) and natural pasture (the whole plant), the action of the enzyme complex enhances methane production. However, when replicating these experiments at the field level in Cuba, the results were not satisfactory. In the case of the experiments carried out in Cuba, a swine inoculum was used, but perhaps the limiting factor of the low yields obtained by the biomass investigated was the size of the particles. They were not sufficiently crushed due to the damage in the mill used and that could affect the interaction enzyme biomass. According to ^{Brulé (2014)}, experiments in the enzymatic hydrolysis stage show low efficiency when enzymes are added to agricultural substrates. The author concluded that the efficiency of enzymes could be favored by the low content of recalcitrant fibers and lignin, low pH and temperature. In addition, he refers that, to achieve a positive effect of the addition of enzymes in productive practice, anaerobic bioreactors must have high organic load (OLR), low hydraulic residence time (HRT) and the substrates must have a mixture of energy crops. Therefore, these results are in contradiction with what was reported by ^{Brulé et al. (2011)}. The results of this investigation indicate a positive effect with the addition of the enzyme ZY maXX XL 200 to the substrates evaluated, and when an appropriate inoculum density is used. The potential of the ZY maXX XL 200 enzyme, to favor the hydrolysis process, which is the limiting stage in anaerobic digestion of these types of waste, should be highlighted. When the hydrolysis is improved, the anaerobic treatment of these wastes is indirectly improved and, therefore, the production of biogas.

The cost of the ZY maXX XL 200 enzyme is $ 64.00 € / kg, and taking into account the small amounts of enzymes used in this experiment (100 µl / syringes per 0.350 mg of substrate evaluated or 0.01 L / 3.5 kg), an approximate cost of € 192 per ton of treated substrate can be inferred, demonstrating the economic feasibility of its use. In addition, the specific values of methane obtaining ​​would reach values ​​from a maximum of 0.047 CH4 / kg VS (mulatto pasture) to a minimum of 0.024 CH4 / kg VS (CT-115) increments, compared with the same substrates without pre-treatments.

The results obtained regarding the evolution of the pH are shown in Figure 2

FIGURE 2 pH evolution (CT-115, mulatto pasture and natural pasture), analyzed under field conditions with enzymes pre-treatments and pig inoculum. 

Regarding the evolution of the pH, in Figure 2, it was seen that the results obtained in the biomass evaluated, showed that all the substrates at the end of the anaerobic biodigestion cycle presented pH values above 7. Therefore, the enzyme ZY maXX XL 200 allows maintaining adequate conditions for a good degradation activity of the substrates in the pH ranges between 6 and 8, which agrees with the results raised by ^{Vintiloiu et al. (2009)}.

Table 3 shows a summary of the average values obtained in each substrate analyzed for biogas and methane yield, respectively, on laboratory scale.

TABLE 3 Average values obtained from specific biogas and methane yield 

Substrate	l/kgFM (m^3/kg VS)	l/kgSV (m^3 CH4/kg VS)
	Specific biogas yield	Specific methane yield
Mulatto pasture	(0,485±0,020 m^3/kg VS)	(0,268±0,01 m^3 CH4/kg VS)
Natural pasture	(0,452±0,041 m^3/kg VS)	(0,254±0,023 m^3 CH4/kgVS)
CT-115 pasture	(0,461±0,026 m^3/kg VS)	(0,258±0,018 m^3 CH4/kg VS)

From the analysis in Table 3, it was observed that the maximum values of the specific biogas yield were obtained in the mulatto pasture (0.485 m³ / kg VS). Meanwhile, the minimum value was obtained with natural pasture (0.452 m³ / kg VS). Similarly, the maximum specific yield values of methane were observed with the mulatto pasture (0.268 m³ CH₄ / kg VS) and the minimum values obtained coincided with the natural pasture (0.254 m³ CH₄ / kg VS). These results achieved on the laboratory level differ from those obtained by ^{Martinez et al. (2014)}, which studied other biomass valued without the addition of enzymes on laboratory scale; as well as from those obtained by ^{Martínez et al. (2015)}, where they used a chemical-thermal pre-treatment for those same substrates.

The results of the statistical analysis of the specific methane yield variable (m³ CH₄ / kg VS) in the evaluated treatments are presented in Figure 3.

FIGURE 3 Tukey test in the variable specific yield of methane (m3 CH4 / kg VS) in the evaluated treatments. 

From the analysis of Figure 3, it was observed that the highest values of the specific yield of methane (m³ CH₄ / kg VS), are obtained with the substrates treated with enzymes with respect to the reference substrate (Standard Hay) presenting significant differences with respect to substrates not treated with enzymes with the exception of CT-115.

CONCLUSIONS

• In all cases, significant increases in the specific methane yield were achieved when a pre-treatment with the enzyme ZY maXX XL 200 was applied to the substrates. The best results took place with mulatto pasture (0.268 m³ CH₄ / kg VS ), followed by CT-115 pasture (0.258 m³ CH₄ / kg VS) and culminating with natural pasture (0.254 m³ CH₄ / kg VS). Significant increases were obtained when the pre-treatment with enzymes and bovine inoculum were used vs. those without them.

• The results achieved on the field level in Cuba with the same biomass evaluated in Germany using pig inoculum were not favorable; however, these results should not be taken as conclusive.

• The pre-treatment with the enzyme ZY maXX XL 200 did not affect the critical pH value in the pre-treated biomass, both in the case of the use of bovine inoculum and porcine inoculum.

ACKNOWLEDGEMENTS

To the national Project of CUBAENERGIA. Study of basic and special pre-treatments to improve the production and quality of the biogas. Code: 9917; To the University of Hohenheim. Germany.

REFERENCES

BRULÉ, M.: The effect of enzyme additives on the anaerobic digestion of energy crops, [en línea], Institut für Agrartechnik, Landesanstalt für Agrartechnik und Bioenergie, Fakultät Agrarwissenschaften, PhD. Thesis, Universität Hohenheim, Germany, 180 p., 2014, Disponible en: http://opus.uni-hohenheim.de/volltexte/2014/1030/ , [Consulta: 15 de diciembre de 2014].

BRULÉ, M.; VOGTHERR, J.; LEMMER, A.; OECHSNER, H.; JUNGBLUTH, T.: “Effect of enzyme addition on the methane yields of effluents from a full-scale biogas plant.”, Landtechnik, 66(1): 50-52, 2011, ISSN: 0023-8082.

FORSTER-CARNEIRO, T.; ISAAC, R.; PÉREZ, M.; SCHVARTZ, C.Anaerobic Digestion: Pretreatment of Substrates. En: Ackmez Mudhoo. Biogas production. Pretreatment methods in anaerobic digestion. Edit. Scrivener-Wiley, 2012. p.1-20.

KURAKAKE, M.; IDE, N.; KOMAKI, T.: “Biological pretreatment with two bacterial strains for enzymatic hydrolysis of office paper”, Current microbiology, 54(6): 424-428, 2007, ISSN: 0343-8651.

MARTÍNEZ, C.; OECHSNER, H.; REINHARDT, A.; GARCÍA, Y.; LÓPEZ, L.: “Studies of chemical-thermal pre-treatment in biomass to use for biogas production in Cuba”, Journal of Basic and Applied Research International, 14(3): 215-224, 2015.

MARTÍNEZ, H.C.; GARCÍA, L.Y.: “Utilización de pre-tratamientos básicos y específicos para la producción de biogás. Revisión y análisis”, Revista Ciencias Técnicas Agropecuarias, 25(3): 81-92, 2016, ISSN: 1010-2760, E-ISSN: 2071-0054.

MARTINEZ, H.C.; OECHSNER, H.; BRULÉ, M.; MARAÑON, M.E.: “Estudio de algunas propiedades físico-mecánicas y químicas de residuos orgánicos a utilizar en la producción de biogás en Cuba”, Revista Ciencias Técnicas Agropecuarias, 23(2): 63-69, 2014, ISSN: 1010-2760, E-ISSN: 2071-0054.

NC 74-22: 85: Determinación de la humedad inicial y de la materia seca, Inst. Oficina Nacional de Normalización (NC), La Habana, Cuba, Vig de 1985.

NC 74-30: 85: Determinación del contenido de cenizas, Inst. Oficina Nacional de Normalización (NC), La Habana, Cuba, Vig de 1985.

TAHERZADEH, M.J.; KARIMI, K.: “Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review”, International journal of molecular sciences, 9(9): 1621-1651, 2008, ISSN: 1422-0067, DOI: 10.3390/ijms9091621.

TANIGUCHI, M.; SUZUKI, H.; WATANABE, D.; SAKAI, K.; HOSHINO, K.; TANAKA, T.: “Evaluation of pretreatment with Pleurotus ostreatus for enzymatic hydrolysis of rice straw”, Journal of bioscience and bioengineering, 100(6): 637-643, 2005, ISSN: 1389-1723, DOI: https://doi.org/10.1263/jbb.100.637.

VDI-4630.: Fermentation of organic material characterization of substrate, sampling collection of material data, fermentation tests ,[en línea], no. 4630, no. 4630, Inst. Inst. VDI-Richtlinien, Berlin, Vig de 2006.

VINTILOIU, A.; BRULÉ, M.; LEMMER, A.; OECHSNER, H.; JUNGBLUTH, T.; JURCOANE, S.; ISRAEL, R.F.: “Influence of temperature and pH value on enzyme activity in the biogas process.”, Landtechnik, 64(1): 22-24, 2009, ISSN: 0023-8082.

ZHONG, W.; ZHANG, Z.; QIAO, W.; FU, P.; LIU, M.: “RETRACTED: Comparison of chemical and biological pretreatment of corn straw for biogas production by anaerobic digestion”, Renewable Energy, 36(6): 1875-1879, 2011, ISSN: 0960-1481, DOI: https://doi.org/10.1016/j.renene.2010.12.020.

⁷The mention of trademarks of specific equipment, instruments or materials is for identification purposes, there being no promotional commitment in relation to them, neither by the authors nor by the publisher.

Received: February 15, 2018; Accepted: December 19, 2019

^* Author for correspondence: Carlos M. Martínez Hernández, e-mail: [e-mail]

Carlos M. Martínez Hernández, Profesor Titular, Universidad Central “Marta Abreu”de las Villas. Carretera a Camajuaní Km.5.5. CP: 54830. Santa Clara. Villa Clara. Cuba, e-mail: [e-mail]

Hans Oechsner, Professor, Universidad de Hohenheim, Landensanstalt fur Agrartechnik und Bioenergie (740). Garbenstrasse 9, Stuttgart. Germany, e-mail: [e-mail]

Annett Reinhard, Universidad de Hohenheim, Landensanstalt fur Agrartechnik und Bioenergie (740). Garbenstrasse 9. Stuttgart. Germany, e-mail: [e-mail]

Yaser García López, Profesor, Universidad Central “Marta Abreu”de las Villas. Carretera a Camajuaní km.5.5. CP: 54830. Santa Clara. Villa Clara. Cuba, e-mail:

Arnaldo Martínez Hernández, Profesor, Dpto de Ciencias Naturales. Universidad Central “Marta Abreu”de las Villas. CP: 50300. Santa Clara. Villa Clara. Cuba, e-mail: [e-mail]

The authors of this work declare no conflict of interests.