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INTRODUCTION
At the present time numerous countries look for solving their energy problems with the use of renewable energy sources. Cuba does not escape to this problem and in the last years the country has carried out an enormous effort to revert its energy matrix, which depends in more than 70% of the electric power production starting from the generation in plants of great size denominated "thermoelectric" and to smaller scale in the calls "power unit". In both, the electricity generation is supported on the base of the consumption of fossil fuel (gasoil and petroleum).
For such a reason, in the Plan of National Development up to 2030, it is foreseen that the country generates around 30% of electricity it consumes, using renewable sources of energy (solar panels, photovoltaic panels, wind power, mini hydraulic power and biogas). In this context, Cuban Universities, as well as several investigation national centers, are projected to the study and investigation of these renewable sources and their optimization.
Therefore, this work has as objective to present the HBT methodology used in Germany to study and value the methane potential of different agricultural substrates in order to transfer the best investigative results obtained toward the production. The HBT methodology presented could be analyzed as a road for its introduction in Cuban biogas laboratories or in biogas laboratories located in the same geographical area.
MATERIALS AND METHODS
Anaerobic Digestion Test. Objectives of the Test
Anaerobic digestion assays were conducted under laboratory conditions using the HBT process (Hohenheim Biogas-yield Test), which is a batch (discontinuous) process. The substrate was digested under mesophilic conditions at constant temperature (37°C) and the retention time was 35 days. Continuous mixing was applied to the digesters.
The aim of developing the HBT was to simplify the assessment of methane potential of a substrate and to run a great number of digesters simultaneously. Further objectives were to reduce the lab tests required as well as the supervision during the tests. The objective of the new procedure was to set it up in such a way that it could be carried out with ordinary commercial laboratory equipment (Shumi, 2008).
Standard Methodology
The BMP-assays were performed according to the German standard VDI 4630 (2006) “Fermentation of organic materials”, developed specifically to assess the methane production during anaerobic digestion. Also International standards currently in force, such as DIN 38424-8 for sewage sludge, ASTM and ISO 14853, were considered. However, the latter standards were developed mainly for the purpose of toxicity tests, not methane yield determination. Moreover, they involve complex methodologies, such as the addition of a wide range of nutrients and micronutrients to sustain anaerobic digestion process, and are not easily practicable (Müller, 2004).
The most important recommendations according to VDI 4630 (VDI 4630, 2006) standard, are that the ratio of weight of substrate VS to weight of inoculum VS has to be set below 0.5 to ensure a stable anaerobic digestion, and the ratio of substrate gas production to the total gas production should be above 0.8.
Equipment
The HBT system allows the investigation of up to 130 single samples at once. The HBT assay is derived from the “Hohenheim Feed value Test” (HFT) developed in Germany (1986) for evaluating the nutritive value of cattle feed with rumen fluid as an inoculum. Its adaptation for biogas applications has been described by Helffrich and Oechsner (Helffrich and Oechsner, 2003) and patented (Helffrich et al., 2005).
Each prepared mixture (substrate + inoculum) is brought into a syringe of 100 mL capacity with a single rubber pipe and a fastening clip upon it as a gas outlet (Figures 1 and 2). The remaining air is removed by pushing the plunger inside the syringe, and anaerobic conditions are reached. The vacuum space between the plunger and the syringe is waterproofed by means of fatty silicon paste.
The syringes are then placed horizontally into a rotating support, which is itself placed in an incubator (Figures 1 and 2), where mesophilic temperature condition (37°C) is maintained. The continuous rotation of the support ensures the thorough mixing of the substrate (Eyler and Brulé, 2010).
The gas produced remains inside the syringe and pushes the plunger away. The gas volume as well as its methane content should be measured each time it exceeds 20 mL. The gas can be measured until its amount reaches 70 mL. This provides a safety interval because some substrates are producing gas at a faster rate than others and because there are always risks of delaying gas measurements. A laser alarm system warns the research staff when the gas volume exceeds 70 mL (Brulé, 2005).
The electronic methane sensor (Figure 8 C) is equipped with a phosphorus pentoxide filter in order to remove water vapor contained in the gas. This water has a corrosive effect in combination with hydrogen sulphide (H2S) when it condenses on metallic parts. H2S gives up sulphuric acid (H2SO4), and the acid aqueous phase produces a corroding effect. The phosphorus pentoxide product (SICAPENT®) has a color indicator which changes color when the salt becomes saturated with water (Brulé, 2005).
During the total fermentation period of 35 days, the gas volume produced from crop substrates is usually measured 10 to 20 times. The pre-digested inoculum is measured only twice because of its very low gas production (Brulé, 2005).
Figure 1 presents some particulars of the group of devices and equipment used in the methodology HBT.
Other particulars of the system HBT is shown in Figure 2.
FIGURE 2 A. Incubator with rotating unit bearing syringes. B. Syringe containing biogas and substrate. C. Methane measurement device (Shumi, 2008).
Substrate Preparation and Feeding
As recommended by VDI 4630 standard, the particle size of the substrate has to be smaller than 10 mm. The substrates used for the tests were chopped at harvest, and passed through a laboratory mixer prior to the test.
Four combinations involving each two different energy crops were chosen to be investigated in different mixture ratios using the HBT process (Table 1). In all the cases the duration of the process was 35 days.
TABLE 1 Experimental design of anaerobic digestion tests
| First crop | Second crop | Mixture ratios |
|---|---|---|
| Perennial ryegrass | Cup plant | 100/0 - 75/25 - 50/50 - 25/75 - [0/100] |
| Corn | Amaranth | 100/0 - 75/25 - 50/50 - 25/75 - 0/100 |
| Corn | Triticale | 100/0 - 75/25 - 50/50 - 25/75 - 0/100 |
| Corn | Perennial ryegrass | 100/0 - 75/25 - 50/50 - 25/75 - 0/100 |
Mixture ratio in parenthesis could not be tested.
Table 2. Shows some examples of mixture ratios and the substrate weights used for the anaerobic digestion test.
Inoculum Preparation and Feeding
The standard inoculum of the University of Hohenheim was used for the trials. The inoculum was produced at the University in a laboratory reactor. It was fed daily with a mixture of predigested dairy manure, maize silage, cereals, rapeseed oil and soybean extract. Predigested manure was taken from an on-farm biogas plant. The C: N ratio of the feed mixture was 27:1 and the organic loading rate of the reactor was 0.5 kg VS/m3/day. This particular procedure was aimed at developing an adapted bacterial population while ensuring a sufficiently low biogas production from the inoculum. Prior to use in HBT-digesters, the inoculum was passed through a 1 mm sieve and coarse material was removed (Brulé, 2005).
In the experiment, 30 g of inoculum were added into each digester, together with the substrate. The role of the inoculum was to provide bacteria, a liquid medium and a buffer effect from carbonates/bicarbonates and ammonia, all of them being necessary for a balanced process.
Control Variants
Control variants were run as specified in VDI 4630 standard VDI 4630 (2006). Other 3 digesters were fed with inoculum only to measure its methane production (control variant). In other 3 digesters inoculum was brought together with one substrate of defined composition and known methane production (substrate standard), in order to check the repeatability of the assay. Here microcrystalline cellulose (250 mg) was used as a substrate standard (AVICEL, MERCK).
Each substrate variant was fed in 3 to 5 digesters, which was considered sufficient to obtain accurate data of the specific methane yield.
Gas Measurement
According to VDI 4630 (2006) standard, the pressure of the generated gas has to be as low as possible so as to minimize gas losses. This recommendation for gas measurement goes against the mainstream of using pressure bottles and pressure transducers (Angelidaki and Sanders, 2004) and favors the use of low pressure systems, such as the eudiometer equipment. Pressure transducers may suffer interferences from temperature variations (Rozzi and Remigi, 2004). The HBT process may be considered as a low-pressure system, since the fluid characteristics of the silicone sealing applied allows an equilibrium at room temperature and normal pressure conditions (Brulé, 2010).
In order to perform a gas measurement, the operator first reads the gas volume on the scale of the syringe. Then, the operator places the syringe vertically, the gas being on the top and the substrate remaining on the bottom. After connecting the exhaust rubber pipe of the syringe to an electronic methane sensor, the operator opens the fastening clip of the rubber pipe to let the gas out and pushes the plunger upwards, letting the gas pass into the electronic device. The value of the methane content provided by the device and the remaining volume of the substrate in the digester are noted down. For each gas measurement, the date, time, exact temperature of the incubator and room air pressure are also recorded in order to calculate the gas production under normal conditions (i.e. at 0°C and 1013.25 hPa) (Brulé, 2005).
Duration of the Experiment
The experiments lasted about 35 days. This duration was applied as a final point, but it does not necessarily imply that the maximal biogas yield can be reached to the end of the experiment. According to VDI 4630 (2006) standard, the experiments can be terminated when the daily biogas production drops below 1% of the total cumulated biogas production. In this study, it was determined that applying the standard assay duration of 35 days allowed fulfilling this criterion.
Calculation of Biogas Yield
According to VDI 4630 (2006) standard, the formula to calculate the volume of dry biogas (corrected from the water vapor) to normal conditions (temperature of 0°C, pressure of 1013, 25 hPa) is as follows:
Where:
V N |
Volume of the dry gas to normalized conditions (L) |
V R |
Read-off value of the biogas volume (L) |
P |
Pressure of the gas at the time of the reading (hPa) |
P w |
Water vapor pressure in biogas (hPa) |
T 0 |
Normal temperature (273 K) |
P 0 |
Normal pressure (1013, 25 hPa) |
T R |
Temperature of biogas (K) |
The water vapor pressure in biogas was estimated to be 58 hPa at 35°C.
Anaerobic Digestion Test
The methane yield of a mixture was calculated according to several input variables, which should be defined for each energy crop: specific methane yield, volatile solids content and dry mass yield per hectare obtained from the field. Then, knowing the weight ratios of each plant in the mixture, the Excel tool can be used to evaluate the methane yield of the chosen mixture.
The values for the specific methane yields given in the literature vary widely depending on the references. For this reason, mixtures and single crops alone were investigated by means of a laboratory test, in order to validate the values for the specific methane yields.
RESULTS AND DISCUSSION
The results reached in a certain experiment performed in the laboratory of Biogas of the University of Hohenheim are presented like an example. These preliminary results are shown in Figure 3. The curves represent specific methane yields for the different ratios of the mixture maize / perennial ryegrass. The methane production increased over the time, but after only 18 days of experiment no conclusion can be drawn about the final yield. Only the final methane yields which were reached after 35 days of experiment could be used as input variables for the model.
Anaerobic digestion assays carried out in the laboratory should provide the best conditions for the biogas process in order to obtain the highest reachable methane yield. Since the conditions for the biological process were optimized, the assay cannot account for synergetic or inhibitory effects which are common in full-scale biogas plants, especially, fiber content and C: N ratios which are output parameters in the model do not play any role in the anaerobic digestion assay. The high amount of inoculum used should buffer against high fibre contents as well as nutrient unbalance.
Therefore, contrary to practice, no synergetic or antagonistic effect is expected: maximal methane yields should be reached in any case, regardless mixture C: N ratio and fiber contents. The investigation of substrate mixtures will serve to confirm this hypothesis. Current results suggest that there is no inhibition associated to a particular mixture: all curves for the mixtures are similar. Surprisingly, the energy crops alone, Corn (Maize) 100% and Perennial ryegrass 100%, show different patterns as compared to the mixtures. These results are very surprising and the experimental protocols should be checked carefully.
FIGURE 3 Curves of the specific methane yield of maize / perennial ryegrass. Search: Biogas lab. Hohenheim University.
In the case of Cuba, this methodology is not very well-known and there are references on the use of this type of methodology only in the works reported by Martínez & García (2016); Martínez et al. (2017) & Martínez et al. (2020), however, it would be profitable to disseminate its use in the country, due to the saving of time, materials and financial resources in the valuation of the methane potential of different agricultural substrates that can be used as raw material for the methane production. Authors of this work are not sure that the methodology HBT has wide use to international scale outside of the European Union (Amon et al., 2001; Amon et al., 2006; Ktbl, 2006; Landesamt für Verbraucherschutz, 2007; Mähnert et al., 2002; Moletta, 2008 ; Van, Robertson, Lewi, 1991; Vindiš et al., 2010; Weiland, 2010). It should be stood out that the work with laboratory digesters of different volumes (2 l up to 50 l) is very common in most of the biogas laboratories to global scale. In the case of the studies with laboratory biodigesters, it can be valued only one substrate or the mixture of some agricultural substrates in an experimental run, unless it has a battery of lab biodigesters, which impulses the process and increases the working expenses, material and financial resources, obtaining the same results that are obtained with the HBT methodology. For such a reason, knowing that the methodology HBT is mature, simple and convincing, its use is recommended keeping in mind the advantages enunciated previously. On the other hand, the transferability of the results achieved to laboratory scale to real scale has been demonstrated in Germany, where more than 40 laboratories exist and use this methodology) (Oechsner et al., 2020).
CONCLUSIONS
The work shows a simple and reliable methodology used to laboratory scale with its proven transferability to real scale for the valuation of different agricultural substrates that can be used as raw material for the obtaining the methane potential from substrates of agricultural or animal origin. Its analysis and valuation are suggested for its possible introduction in the Cuban biogas lab.
