**2. Anaerobic digestion of maize silage in laboratory conditions**

Within the laboratory experiments on anaerobic processing of maize silage, tests of the maize silage biogas potential were carried out, and a long-term operation of a biogas production model was monitored. The aim of these tests was to determine the specific biogas production.

## **2.1. Test of maize silage biomethane potential**

/kg

/kg of

/ha (hybrid maize,

from energy crops. This option has gained wide application in particular in connection with government support of the electricity price produced from biogas in many countries of the EU. In addition, growing and utilization of energetic crops for biogas production is one of the alternatives of agriculture production diversification which can significantly improve farm economics. Energy from biogas produced by anaerobic digestion of energetic crops can be utilized to improve the energetic balance of a farm as excess energy can be sold (e.g. to the electric grid). Maize in the form of silage provides high yields (10–30 t of total solids—TS per hectare [1–3]) and is thus a suitable energetic crop for biogas production. More than 17,000 biogas plants, mostly using maize silage as the main substrate, are in operation in Europe; for example, in Germany, more than 8000 biogas plants have been in operation by the end of 2015 with the plant biomass utilization of more than 52 mass% and of livestock excrements of 43 mass% [4]. The rest are industrial and agro- and food processing waste as well as municipal biowaste. Advantages of plant biomass utilization are even pronounced by the fact that 52 mass% of the total substrates processed in biogas plants result in a 79% energy production. Maize silage represents 73 mass% of the plant biomass processed in the biogas plants, while the energy represents 72% of the total energy production. Thus, in 2014, 56.88% of energy produced by biogas plants in Germany originated from maize silage [4]. Even though no precise information on the species composition of the biogas plant substrates in other countries of the European Union is available, it is clear that the main substrate is maize silage. However, only little information on its anaerobic digestion is provided in literature. Generally, it can be stated that studies on the anaerobic digestion of fresh and ensiled materials did not show any significant differences between the biogas production from these materials [5, 6]. Concerning the anaerobic digestion, the main advantage of ensiling is the conservation of plant substrates to enable biogas production for the whole year. Zauner and Kuntzel [7] present anaerobic processing of maize silage in their work achieving methane production of 0.270–0.289 m3

of TS in a laboratory batch reactor. The production was somehow lower, 0.181–0.184 m3

0.206–0.283 Nm3

174 Advances in Silage Production and Utilization

0.282–0.419 Nm3

to 8530 Nm3

TS, in continuous laboratory reactors. Amon et al. [2] studied the biogas production from maize and clover grass in more detail. They focused on the biogas production of various species in different stages (milk, wax, and full ripeness). Also the influence of ensiling and drying on the methane production was studied. Various species had different optimal harvesting time in different ripeness stages. Specific methane production was in the range of

digestion in mesophilic conditions (40°C) for 60 days. Some maize varieties showed minimum difference in the methane production considering the ripeness stage and some showed a difference of more than 25% (variety Saxxo, wax ripeness) [2]. Specific methane production of

effect of maize composition and ripeness stage on the methane yield. Specific methane production from hybrids with late ripeness increased with the higher date of sampling more significantly than from climatically adapted "medium-early" hybrids, which reached the maximum methane production more quickly. These results are comparable with those

provided by other authors [3, 9–14]. In [13], maximum yield of 9440.6 Nm3

/kg of the volatile solids—VS and the methane yield was in the range of 5300

/kg of the VS was achieved in the work of Schittenhelm [8], who studied the

/ha of the VS. These results were obtained by the batch tests of the anaerobic

It is necessary to emphasize that the tests of biomethane potential have only an informational character and the specific methane production reached by long-term operation of anaerobic digestion of biologically degradable substrates can differ considerably. Single biomethane potential test results are significantly affected by the anaerobic sludge used as inoculum. Used anaerobic sludge is not usually adapted to the substrate degradation and during batch test the adaptation is not carried out. Therefore, the biomethane potential test provides a value lower than that obtained by long-term anaerobic digestion, when the adaptation of the anaerobic sludge to the used substrate and thus deeper anaerobic digestion of the substrate can take place. If the substrate contains a toxic or inhibitory substance, its influence might not be demonstrated during the biomethane potential test, due to the sufficient dilution of the substrate by the anaerobic sludge used for inoculation, for example, in substrates with high nitrogen or sulfur content. In long-term anaerobic digestion process, when the substrate is repeatedly supplied to the reactor, nitrogen or sulfur can accumulate in the reactor, and ammonia or sulfide inhibition of anaerobic processes can occur gradually. However, the biomethane potential test is a suitable tool for the primary evaluation of anaerobic digestion of a substrate and the possible biogas production.

Maize silage produced at the STIFI farm in Hurbanovo was used in the biomethane potential test without particle size adjustment. The particle size was given by the harvesting machine as up to 5 cm in length. Silage was made in the traditional way. Harvesting took place at the TS of the green maize about 30%. After the cropping, green maize was compacted by bulldozer in the silage pit with dimensions of 22 m × 75 m × 5 m. For tests, the silage after two month of ensiling was used. Content of TS of the used silage represented 35% with the VS content of 95.8%. Value of pH of maize silage water leachate (100 g of silage in 400 ml of tap water) was 3.7. Anaerobically stabilized sludge from the municipal wastewater treatment plant in Devínska Nová Ves (total suspended solids— TS of 37.23 g/L and volatile suspended solids— VS of 20.74 g/L) in the volume of 0.5 l and 7 g of fresh silage was used for the tests. The sludge mixture was completed to the total volume of 1 l with tap water. To determine the biogas production from the anaerobically stabilized sludge, a blank test was done. The tests were carried out in the mesophilic temperature regime (35°C) in three repetitions. The biomethane potential results are presented in **Figure 1**.

carried out applying standard methods [22]. The analysis of VFA was done employing the method introduced by Kapp [23]. To determine the biogas composition (methane, CO2, H2,

Operation of the laboratory reactor for anaerobic digestion of maize silage started at the organic

of the anaerobic reactor are provided in **Table 1**. **Figure 2** shows the course of the specific biogas production per kg of added VS and the cumulative biogas production in the anaerobic reactor with gradual increase of OLR in the reactor. OLR increased from 1.68 to 6.71 kg/(m3

**Table 1**. Average specific biogas production at individual OLR values was in the range of 0.195–

/d). The course of COD and VFA is shown in **Figure 3** and that of pH and NH4-N in **Figure 4**. Instability of the processes was demonstrated by the decrease of pH and the increase of COD and VFA concentration, especially after the increase of OLR in the reactor (**Figures 2**

per kg of VS. Maximum specific biogas production was achieved at the OLR of 5.03 kg/

/d). The course of loading doses and the achieved parameters

**Dose of silage (VS)**

**[g/d]**

/d)—

177

**Specific biogas production**

**/kg]**

**(VS) [m3**

Maize Silage as Substrate for Biogas Production

http://dx.doi.org/10.5772/64378

and H2S), the apparatus GA 2000 Plus (Geotechnical Instruments, UK) was used.

**Day of operation Dose of silage (raw**

**material) [g/d]**

1.68 0–20 20 6.71 0.195 2.52 21–40 30 10.06 0.230 3.36 41–80 40 13.42 0.430 4.19 81–120 50 16.77 0.530 5.03 121–220 60 20.12 0.655 6.71 220–300 80 26.83 0.420

**Table 1.** Anaerobic processing of maize silage—operation parameters of the anaerobic reactor.

**Figure 2.** Specific and cumulative biogas production in the anaerobic reactor.

loading rate (OLR) of 1.68 kg/(m3

0.655 m3

and **3**).

**Organic loading rate (VS) [kg/(m3**

 **d)]**

(m3

**Figure 1.** Test of biomethane potential of maize silage.

From the test results follows that 233 ml of methane per gram of TS (243 ml per gram of VS) respectively 0.206 Nm3 /kg of TS (0.215 Nm3 /kg of VS) were produced. It is in agreement with results provided by Amon et al. [2].

#### **2.2. Long-term anaerobic digestion of maize silage in the laboratory conditions**

Long-term maize silage processing was carried out in a mixed laboratory anaerobic reactor with the volume of 4 l. The reactor was filled to the half of its volume with the anaerobically stabilized sludge used for biomethane potential tests (TS of 37.2 g/L with VS of 55.7%) and was filled to the total volume of 4 l with tap water. Silage was processed in its raw form without any pre-treatment, that is, as taken from the silage pits in STIFI Hurbanovo, and stored at 4°C. The silage was loaded once a day into the laboratory model operated at 35°C. In the filtered samples of sludge water, parameters as chemical oxygen demand (COD), volatile fatty acids (VFA), ammonia nitrogen (NH4-N), and pH were determined. Also the concentration of suspended solids and biogas production were monitored in the reactor. All analyses were carried out applying standard methods [22]. The analysis of VFA was done employing the method introduced by Kapp [23]. To determine the biogas composition (methane, CO2, H2, and H2S), the apparatus GA 2000 Plus (Geotechnical Instruments, UK) was used.

TS of the green maize about 30%. After the cropping, green maize was compacted by bulldozer in the silage pit with dimensions of 22 m × 75 m × 5 m. For tests, the silage after two month of ensiling was used. Content of TS of the used silage represented 35% with the VS content of 95.8%. Value of pH of maize silage water leachate (100 g of silage in 400 ml of tap water) was 3.7. Anaerobically stabilized sludge from the municipal wastewater treatment plant in Devínska Nová Ves (total suspended solids— TS of 37.23 g/L and volatile suspended solids— VS of 20.74 g/L) in the volume of 0.5 l and 7 g of fresh silage was used for the tests. The sludge mixture was completed to the total volume of 1 l with tap water. To determine the biogas production from the anaerobically stabilized sludge, a blank test was done. The tests were carried out in the mesophilic temperature regime (35°C) in three repetitions. The biomethane

From the test results follows that 233 ml of methane per gram of TS (243 ml per gram of VS)

Long-term maize silage processing was carried out in a mixed laboratory anaerobic reactor with the volume of 4 l. The reactor was filled to the half of its volume with the anaerobically stabilized sludge used for biomethane potential tests (TS of 37.2 g/L with VS of 55.7%) and was filled to the total volume of 4 l with tap water. Silage was processed in its raw form without any pre-treatment, that is, as taken from the silage pits in STIFI Hurbanovo, and stored at 4°C. The silage was loaded once a day into the laboratory model operated at 35°C. In the filtered samples of sludge water, parameters as chemical oxygen demand (COD), volatile fatty acids (VFA), ammonia nitrogen (NH4-N), and pH were determined. Also the concentration of suspended solids and biogas production were monitored in the reactor. All analyses were

**2.2. Long-term anaerobic digestion of maize silage in the laboratory conditions**

/kg of VS) were produced. It is in agreement with

potential results are presented in **Figure 1**.

176 Advances in Silage Production and Utilization

**Figure 1.** Test of biomethane potential of maize silage.

results provided by Amon et al. [2].

/kg of TS (0.215 Nm3

respectively 0.206 Nm3

Operation of the laboratory reactor for anaerobic digestion of maize silage started at the organic loading rate (OLR) of 1.68 kg/(m3 /d). The course of loading doses and the achieved parameters of the anaerobic reactor are provided in **Table 1**. **Figure 2** shows the course of the specific biogas production per kg of added VS and the cumulative biogas production in the anaerobic reactor with gradual increase of OLR in the reactor. OLR increased from 1.68 to 6.71 kg/(m3 /d)— **Table 1**. Average specific biogas production at individual OLR values was in the range of 0.195– 0.655 m3 per kg of VS. Maximum specific biogas production was achieved at the OLR of 5.03 kg/ (m3 /d). The course of COD and VFA is shown in **Figure 3** and that of pH and NH4-N in **Figure 4**. Instability of the processes was demonstrated by the decrease of pH and the increase of COD and VFA concentration, especially after the increase of OLR in the reactor (**Figures 2** and **3**).


**Table 1.** Anaerobic processing of maize silage—operation parameters of the anaerobic reactor.

**Figure 2.** Specific and cumulative biogas production in the anaerobic reactor.

operation. The average sodium bicarbonate consumption was 0.05 g/g of VS silage. Instability of the anaerobic processing of maize silage is related to it insufficient acid neutralizing capacity (alkalinity) due to the high C/N ratio in this substrate (30–46) [3]. Together with the carbonate

role in the anaerobic processes. Results of long-term anaerobic reactor operation indicate that anaerobic digestion of maize silage as the only substrate requires the presence of alkaline reagents. From a practical point of view and that of nutrients demand, loading of a co-substrate with higher content of nitrogen, for example, sewage sludge, or manure, is required. At OLR

tively. It is thus clear that at this OLR value, the system was permanently overloaded and the COD and VFA values were stabilized at 6000 mg/L and 2800 mg/L, respectively. Also the specific biogas production was considerably lower (0.420 kg/kg of VS) as that at the load of

During the stable operation of the reactor (days 121–220), the average concentration of suspended solids in the anaerobic reactor was 79 g/L. Daily amount of the suspended solids of excess sludge was 3.57 g. At the load of 21 g of TS silage (60 g of fresh silage with the TS content of 35%), the production of excess sludge was 0.17 g pre 1 g of TS, which corresponds to the anaerobic silage digestion degree of 83%. The content of individual biogas components is provided in **Table 2** and the parameters of anaerobic digestion of maize silage obtained from

**Parameter Value**

Suspended solids in reactor [g/L] 79

Specific excess sludge production 0.17 Degradation of TS [%] 83.0

/d)] 5.03

/kg VS] 0.655

/kg VS] 0.316

), ammonia buffer system (NH3/NH4

/d), the COD and VFA values exceeded 18,000 mg/L and 11,000 mg/L, respec-

/d), with the highest specific production of biogas.

+

Maize Silage as Substrate for Biogas Production

/kg of VS). Therefore, the optimal value of OLR has

) also has an important

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179

buffer system (CO2/CO3

been established as 5.03 kg/(m3

of 6.71 kg/(m3

5.03 kg/(m3

OLR (VS) [kg/(m3

Specific biogas production (35°C) [m3

**Table 3.** Parameters of maize silage anaerobic digestion.

Specific methane production [Nm3

2−/HCO3 −

/d) (0.655 kg/kg of VS)(0.655 m3

the laboratory model are summarized in **Table 3**.

**Component Value** CH4 [%] 54.5 CO2 [%] 45.4 H2 [ppm] 5 H2S [ppm] 215

**Table 2.** Composition of biogas produced from maize grains and maize silage.

**Figure 3.** COD and VFA in the laboratory anaerobic reactor.

**Figure 4.** NH4-N and pH in the laboratory anaerobic reactor.

Stabilization of COD and VFA took several days or even weeks depending on the destabilization degree after the silage load increase. At higher loads, the response to increased OLR was stronger and the stabilization period was longer. With the OLR increase, pH decreased below 6.5 (**Figure 3**) and sodium bicarbonate was used to adjust the pH. The pH value had to be adjusted not only after an OLR increase but throughout the anaerobic reactor operation because pH in neutral range is needed for methanogenic microorganisms. In total, ca. 6000 g of VS silage and 100 g of sodium bicarbonate were loaded into the reactor during its 300 day operation. The average sodium bicarbonate consumption was 0.05 g/g of VS silage. Instability of the anaerobic processing of maize silage is related to it insufficient acid neutralizing capacity (alkalinity) due to the high C/N ratio in this substrate (30–46) [3]. Together with the carbonate buffer system (CO2/CO3 2−/HCO3 − ), ammonia buffer system (NH3/NH4 + ) also has an important role in the anaerobic processes. Results of long-term anaerobic reactor operation indicate that anaerobic digestion of maize silage as the only substrate requires the presence of alkaline reagents. From a practical point of view and that of nutrients demand, loading of a co-substrate with higher content of nitrogen, for example, sewage sludge, or manure, is required. At OLR of 6.71 kg/(m3 /d), the COD and VFA values exceeded 18,000 mg/L and 11,000 mg/L, respectively. It is thus clear that at this OLR value, the system was permanently overloaded and the COD and VFA values were stabilized at 6000 mg/L and 2800 mg/L, respectively. Also the specific biogas production was considerably lower (0.420 kg/kg of VS) as that at the load of 5.03 kg/(m3 /d) (0.655 kg/kg of VS)(0.655 m3 /kg of VS). Therefore, the optimal value of OLR has been established as 5.03 kg/(m3 /d), with the highest specific production of biogas.

During the stable operation of the reactor (days 121–220), the average concentration of suspended solids in the anaerobic reactor was 79 g/L. Daily amount of the suspended solids of excess sludge was 3.57 g. At the load of 21 g of TS silage (60 g of fresh silage with the TS content of 35%), the production of excess sludge was 0.17 g pre 1 g of TS, which corresponds to the anaerobic silage digestion degree of 83%. The content of individual biogas components is provided in **Table 2** and the parameters of anaerobic digestion of maize silage obtained from the laboratory model are summarized in **Table 3**.


**Table 2.** Composition of biogas produced from maize grains and maize silage.


**Table 3.** Parameters of maize silage anaerobic digestion.

**Figure 3.** COD and VFA in the laboratory anaerobic reactor.

178 Advances in Silage Production and Utilization

**Figure 4.** NH4-N and pH in the laboratory anaerobic reactor.

Stabilization of COD and VFA took several days or even weeks depending on the destabilization degree after the silage load increase. At higher loads, the response to increased OLR was stronger and the stabilization period was longer. With the OLR increase, pH decreased below 6.5 (**Figure 3**) and sodium bicarbonate was used to adjust the pH. The pH value had to be adjusted not only after an OLR increase but throughout the anaerobic reactor operation because pH in neutral range is needed for methanogenic microorganisms. In total, ca. 6000 g of VS silage and 100 g of sodium bicarbonate were loaded into the reactor during its 300 day Considering that from 1 ha of arable land, 30 t of TS silage (VS of 95%) per annum are obtained, methane production is 9006 Nm3 /ha. For a biogas plant with produced biogas incineration in a cogeneration unit with the electric power of 1 MW (electric energy production efficiency of 35%), the daily maize silage demand represents the area of 0.77 ha. This means that the annual operation of biogas plants needs 8431.5 t of TS silage, grown on 281 ha of arable land.

Conclusions of the anaerobic digestion of maize silage in laboratory conditions:

Biomethane potential tests provided the measured specific methane production of 0.215 Nm3 /kg of VS. For long-term maize silage processing in a mixed laboratory anaerobic reactor, the measured specific methane production was 0.316 Nm3 /kg of VSS. The higher value obtained for long-term reactor operation is due to the adaptation of the anaerobic microorganisms to the maize silage substrate.

Long-term operation of the anaerobic reactor for maize silage processing as the only substrate showed significant instability caused by the low alkalinity of maize silage (high C:N ratio). To stabilize the anaerobic processes, other alkaline reagents or a co-substrate with higher content of nitrogen (sewage sludge or manure) can be used.

Daily operation of a biogas plant with biogas incinerated in a cogeneration unit with the electric power of 1 MW requires the amount of silage from an area of 0.77 ha of arable land.

inoculation of the anaerobic reactor the anaerobically stabilized sludge is used, but the distance to the nearest wastewater plant with anaerobically stabilized sludge was 15 km, and the required amount of sludge could not be provided. Aerobically stabilized sludge from brewery wastewater plant was available for only as far as 2 km from the biogas plant. The amount of aerobically stabilized sludge added to the anaerobic reactor for inoculation before its start-up was 1700 m3 with an average concentration of suspended solids (SS) of 30 g/L. After the inoculation, the reactor was heated to 37°C and gradually loaded with maize silage. During the start-up of the anaerobic reactor, biogas production, pH, VFA, NH4-N, PO4-P, and suspended solids concentration were monitored. The course of these parameters was also monitored during the first

Maize Silage as Substrate for Biogas Production

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181

200 days of the pilot plant operation (**Figures 6**–**10**).

**Figure 6.** Course of silage dose and biogas production during the start-up of the anaerobic reactor.

**Figure 5.** Diagram of biogas plant for anaerobic digestion of the maize silage.
