**4. Influence of different substrates on operation of biogas plant for anaerobic digestion of maize silage as main substrate**

From the beginning of the biogas plant operation, maize silage was used as the main and often the only substrate. Maize silage composition has changed depending on different factors, for example, the maize variety used. One of the most important factors is the ripeness season when maize is harvested for ensiling [3, 8]. In **Table 5**, selected parameters of substrates significantly influencing the biogas plant operation are provided. Except for the maize silage, also meat and bone meal, molasses stillage from bioethanol production—vinasse and a by-product of

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187

**Parameter Maize silage Meat and bone meal Vinasse Crude glycerol**

During the biogas plant operation, other substrates were also processed, for example, rye silage and a mixture of oat and peas, which however did not significantly affect the reactor operation.

Co-substrate loading in the biogas plant had two main reasons: insufficient amount of the main substrate—maize silage, especially in spring; and stabilization of the anaerobic reactor operation, for example, in case of the brewery wastewater plant sludge. The course of various

Loading of various substrates is discussed in relationship with the biogas production and pH changes. The course of biogas production in a biogas plant from the start of its operation is

pH – – 6.15 9.03 Chemical oxygen demand [g/g of TS] 1.22 1.32 – –

Total Kjeldahl nitrogen [% of TS] 0.88 7.90 – – Total Kjeldahl nitrogen [mg/L] – – 19,254 – NH4-N [mg/L] – – 3390 – Total nitrogen [% of TS] 2.69 8.85 – – Total nitrogen [mg/L] – – – 1690 Total phosphorus [mg/L] – – – 192 PO4-P [mg/L] – – 835 – C:N 17.6 4.42 – – Dissolved anorganic salts [mg/L] – – – 5150 Density [g/L] – – – 1080 Lactic acid [%] 1.85 – – – Acetic acid [%] 1.77 – – – TS [%] 30.8 72.5 45.9 – VS of TS [%] 94.1 79.9 71.8 –

Chemical oxygen demand [mg/L] – – 332,930 1,870,000

biodiesel production—crude glycerol characteristics are presented.

**Table 5.** Characteristics of the used substrates.

substrates loading is presented in **Figure 12**.

presented in **Figure 13** and the pH values are shown in **Figure 14**.

Raw materials used for the biogas plant differ not only in their physical properties but also in their composition. From the anaerobic digestion point of view, organic carbon and its proportion to nitrogen are more appreciated. If the organic carbon is in the form of hardly degradable matter and hydrolysis or acidification is required, the effect of substrate dosing will differ from that observed for readily biodegradable matter. If the dosing effect of readily biodegradable substrate on the anaerobic processes is strong and the biogas production increases sharply immediately after the loading, together with other changes in the reaction mixture (pH change, VFA increase, etc.), it is recommended to divide the loadings to as many as possible during the day. Dosing optimization has a positive influence on the processes not only considering the degradation but also concerning the presence of toxic or inhibitory substances.

For efficient anaerobic processes, the balanced substrates composition, especially when considering the macronutrients (nitrogen and phosphorus) content, is also an important factor. Inhibition of ongoing processes can be caused by low nitrogen or phosphorus content, or by high nitrogen content. Optimum COD/N/P ratio for the anaerobic microorganisms growth is in the range of 1000:5:1 for acidified substrate (with low biomass production), and up to 350:5:1 for unacidified substrates (with high biomass production) [29]. For materials processed in biogas plants, COD determination is quite a complex problem; therefore, the ratio of organic carbon to nitrogen (C:N ratio) is usually applied. Generally, it can be stated that for materials with high nitrogen content (blood, meat and bone meal, rapeseed meal, chicken droppings), this ratio is up to 10–15, for materials with medium nitrogen content (maize silage, cereal straw) it is up to approx. 50, and for materials with low nitrogen content (e.g. wood biomass), the C:N ratio is above 50 [30].

At very high C:N ratios, methanogenic microorganisms are not sufficiently supplied with nitrogen to assimilate (growth and propagation) and conditions for organic carbon degradation are not achieved. Or, as in case of maize silage, at low alkalinity of the substrate, pH in the reactor decreases and the process becomes instable. At low values of pH (below 6.5) growth of methanogenic microorganisms is strongly inhibited, because optimal pH for their growth is in the neutral range. However, at very low C/N ratios, nitrogen accumulates in the sludge water in its ammonia form, which can result in a pH increase and anaerobic processes inhibition by undissociated ammonia.

Considering the biomass composition, the presence of sulfur is also important; sulfur in its organic as well as inorganic form is transformed to its reduced forms, mainly to sulfides and hydrogen sulfide, by anaerobic processes. Sulfides present in the anaerobic sludge water are toxic to the methanogenic microorganisms, and hydrogen sulfide causes problems with the biogas incineration in heaters of cogeneration units.

As an example of influent of different substrates dosing on a biogas plant operation, long-term monitoring of the biogas plant in Hurbanovo using maize silage as the main substrate can be provided. Its start-up and trial operation were described above.

From the beginning of the biogas plant operation, maize silage was used as the main and often the only substrate. Maize silage composition has changed depending on different factors, for example, the maize variety used. One of the most important factors is the ripeness season when maize is harvested for ensiling [3, 8]. In **Table 5**, selected parameters of substrates significantly influencing the biogas plant operation are provided. Except for the maize silage, also meat and bone meal, molasses stillage from bioethanol production—vinasse and a by-product of biodiesel production—crude glycerol characteristics are presented.


**Table 5.** Characteristics of the used substrates.

**4. Influence of different substrates on operation of biogas plant for**

the degradation but also concerning the presence of toxic or inhibitory substances.

For efficient anaerobic processes, the balanced substrates composition, especially when considering the macronutrients (nitrogen and phosphorus) content, is also an important factor. Inhibition of ongoing processes can be caused by low nitrogen or phosphorus content, or by high nitrogen content. Optimum COD/N/P ratio for the anaerobic microorganisms growth is in the range of 1000:5:1 for acidified substrate (with low biomass production), and up to 350:5:1 for unacidified substrates (with high biomass production) [29]. For materials processed in biogas plants, COD determination is quite a complex problem; therefore, the ratio of organic carbon to nitrogen (C:N ratio) is usually applied. Generally, it can be stated that for materials with high nitrogen content (blood, meat and bone meal, rapeseed meal, chicken droppings), this ratio is up to 10–15, for materials with medium nitrogen content (maize silage, cereal straw) it is up to approx. 50, and for materials with low nitrogen content (e.g. wood biomass), the C:N

At very high C:N ratios, methanogenic microorganisms are not sufficiently supplied with nitrogen to assimilate (growth and propagation) and conditions for organic carbon degradation are not achieved. Or, as in case of maize silage, at low alkalinity of the substrate, pH in the reactor decreases and the process becomes instable. At low values of pH (below 6.5) growth of methanogenic microorganisms is strongly inhibited, because optimal pH for their growth is in the neutral range. However, at very low C/N ratios, nitrogen accumulates in the sludge water in its ammonia form, which can result in a pH increase and anaerobic processes

Considering the biomass composition, the presence of sulfur is also important; sulfur in its organic as well as inorganic form is transformed to its reduced forms, mainly to sulfides and hydrogen sulfide, by anaerobic processes. Sulfides present in the anaerobic sludge water are toxic to the methanogenic microorganisms, and hydrogen sulfide causes problems with the

As an example of influent of different substrates dosing on a biogas plant operation, long-term monitoring of the biogas plant in Hurbanovo using maize silage as the main substrate can be

Raw materials used for the biogas plant differ not only in their physical properties but also in their composition. From the anaerobic digestion point of view, organic carbon and its proportion to nitrogen are more appreciated. If the organic carbon is in the form of hardly degradable matter and hydrolysis or acidification is required, the effect of substrate dosing will differ from that observed for readily biodegradable matter. If the dosing effect of readily biodegradable substrate on the anaerobic processes is strong and the biogas production increases sharply immediately after the loading, together with other changes in the reaction mixture (pH change, VFA increase, etc.), it is recommended to divide the loadings to as many as possible during the day. Dosing optimization has a positive influence on the processes not only considering

**anaerobic digestion of maize silage as main substrate**

186 Advances in Silage Production and Utilization

ratio is above 50 [30].

inhibition by undissociated ammonia.

biogas incineration in heaters of cogeneration units.

provided. Its start-up and trial operation were described above.

During the biogas plant operation, other substrates were also processed, for example, rye silage and a mixture of oat and peas, which however did not significantly affect the reactor operation.

Co-substrate loading in the biogas plant had two main reasons: insufficient amount of the main substrate—maize silage, especially in spring; and stabilization of the anaerobic reactor operation, for example, in case of the brewery wastewater plant sludge. The course of various substrates loading is presented in **Figure 12**.

Loading of various substrates is discussed in relationship with the biogas production and pH changes. The course of biogas production in a biogas plant from the start of its operation is presented in **Figure 13** and the pH values are shown in **Figure 14**.

**Figure 12.** Course of dosing of various substrates in the biogas plant.

reactor. The main effect of sludge dosing was the increase of N in the system. It is possible, that the other effects of sludge dosing was introducing micronutrients and minerals, but from **Figures 8** and **9** it is obvious that main effect to pH stabilization is the increase of NH4-N

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189

**Figure 14.** Course of pH values in the anaerobic reactor of the biogas plant.

From day 203 of the operation, meat and bone meal was dosed to the reactor, first to increase nitrogen concentration, then also to compensate the lack of maize silage. When the maize silage was not available and only low quality rye silage could be used, the average dose of this silage was 2 t and up to 3.2 t of meat and bone meal. Between days 180 and 250 of the operation, the biogas production achieved its maximum (designed) values. Long-term high loads of meat and bone meal (between days 203–255) had, however, a negative impact on the anaerobic reactor operation; therefore, dosing of this substrate was in day 255 stopped. Meat and bone meal is a substrate with low C:N ratio, of 4.42 (**Table 5**), and thus with high nitrogen content, which resulted in the increase in ammonia nitrogen concentration in the sludge water to above 2800 mg/L and that of pH to above 7.5; the process was probably inhibited by undissociated ammonia in the sludge water. Volatile fatty acids concentration was higher than 7500 mg/L (**Figure 15**) and the biogas production decreased (**Figure 13**). Also the biogas quality was lower, H2S concentration increased from values below 200 ppm to values above 1800 ppm. Loading to the anaerobic reactor was interrupted for two weeks and after the volatile fatty acids concentration decreased below 6000 mg/L, rye silage and maize splits loadings were slowly resumed. Following the initiation of maize silage dosing from the new harvest in about day 300, the reactor operation gradually stabilized and the biogas production almost reached its maximum values. Although the following reactor operation was not quite stable, such

severe conditions as after meat and bone meal dosing did not occur.

concentration.

**Figure 13.** Biogas production in the biogas plant.

Start-up and trial operation were discussed earlier. At this period of the anaerobic reactor operation, the disadvantage of the low alkalinity of maize silage processed as the only substrate was demonstrated. This disadvantage was suppressed by brewery sludge loading to the

**Figure 14.** Course of pH values in the anaerobic reactor of the biogas plant.

**Figure 12.** Course of dosing of various substrates in the biogas plant.

188 Advances in Silage Production and Utilization

**Figure 13.** Biogas production in the biogas plant.

Start-up and trial operation were discussed earlier. At this period of the anaerobic reactor operation, the disadvantage of the low alkalinity of maize silage processed as the only substrate was demonstrated. This disadvantage was suppressed by brewery sludge loading to the reactor. The main effect of sludge dosing was the increase of N in the system. It is possible, that the other effects of sludge dosing was introducing micronutrients and minerals, but from **Figures 8** and **9** it is obvious that main effect to pH stabilization is the increase of NH4-N concentration.

From day 203 of the operation, meat and bone meal was dosed to the reactor, first to increase nitrogen concentration, then also to compensate the lack of maize silage. When the maize silage was not available and only low quality rye silage could be used, the average dose of this silage was 2 t and up to 3.2 t of meat and bone meal. Between days 180 and 250 of the operation, the biogas production achieved its maximum (designed) values. Long-term high loads of meat and bone meal (between days 203–255) had, however, a negative impact on the anaerobic reactor operation; therefore, dosing of this substrate was in day 255 stopped. Meat and bone meal is a substrate with low C:N ratio, of 4.42 (**Table 5**), and thus with high nitrogen content, which resulted in the increase in ammonia nitrogen concentration in the sludge water to above 2800 mg/L and that of pH to above 7.5; the process was probably inhibited by undissociated ammonia in the sludge water. Volatile fatty acids concentration was higher than 7500 mg/L (**Figure 15**) and the biogas production decreased (**Figure 13**). Also the biogas quality was lower, H2S concentration increased from values below 200 ppm to values above 1800 ppm. Loading to the anaerobic reactor was interrupted for two weeks and after the volatile fatty acids concentration decreased below 6000 mg/L, rye silage and maize splits loadings were slowly resumed. Following the initiation of maize silage dosing from the new harvest in about day 300, the reactor operation gradually stabilized and the biogas production almost reached its maximum values. Although the following reactor operation was not quite stable, such severe conditions as after meat and bone meal dosing did not occur.

used was 42.47%) and the daily crude glycerol loading of 683.7 kg. To determine the specific biogas production from crude glycerol, the value of the specific biogas production from maize

substrate) was employed. The average daily amount of biogas produced from maize silage

Biogas produced from crude glycerol represented 14.82% of the total biogas production, while 11.46% of the total TS, 12.1% of VS and only 5.21% of the total mass of the raw materials loaded. At the electrical power output of the cogeneration unit of 300 kW (electrical power output of the cogeneration unit was increased from 276 kW to 300 kW after an agreement with the producer considering the operation experiences), the daily electric energy production from

Amon et al. [32] studied the influence of various loading doses of crude glycerol on the anaerobic digestion of pig manure, maize silage and maize corns. Co-fermentation effect was observed. It means that methane yield of the basic mixture supplemented with glycerol was higher than the combined methane yields of both substrates if digested separately. The cofermentation effect was especially high with glycerin additions of 3–6%. They recommend the

To complete the biogas plant monitoring results obtained for various co-substrates, the course of suspended solids concentration in the anaerobic reactor is provided in **Figure 16**, which shows a gradual increase of this concentration in the period of more than two years. After crude glycerol started to be added to the reactor, the concentration of suspended solids slightly

In our case of long-term crude glycerol loading in the biogas plant, the average glycerol VS addition was 12.1% and the anaerobic digestion process was stable. However, no co-fermen-

Co-substrates used in anaerobic digestion of maize silage as the main substrate can have a

Uncontrolled meat and bone meal loading resulted in a failure of the anaerobic reactor due to the high nitrogen content in this substrate. Its processing together with the maize silage is possible; however, the loading dose has to be regulated considering the ammonia nitrogen

It has been proved that the inhibitory effect of ammonia nitrogen depends on also the course of its increase. While an abrupt increase of the NH4-N concentration to approximately 2800 mg/ L (loading of meat and bone meal) resulted in an inhibition of the anaerobic processes, gradual increase to almost 4500 mg/L (in case of vinasse loading) showed no negative effect on the

Conclusions of the study of various substrates loading on the biogas plant operation:

which corresponds to the specific biogas production of 0.512 m3

crude glycerol was 1067 kWh and almost 15% of silage were saved.

glycerol content of maximum 6% for a stable reactor operation.

decreased, which had a positive effect on the reactor mixing.

positive as well as a negative effect on the biogas plant operation.

concentration and the pH in the anaerobic reactor.

per 1 kg of the maize silage TS, obtained when maize silage was the only

and the average daily amount of biogas produced from crude

,

191

/kg of COD and is in agreement

Maize Silage as Substrate for Biogas Production

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. The specific biogas production per 1 kg of crude glycerol was 0.887 m3

silage (0.66 m3

was calculated as 3485 m3

tation effect was observed.

process.

with the results presented in [31].

glycerol was 606.4 m3

**Figure 15.** Concentration of VFA in filtrated sludge water from the anaerobic reactor in the biogas plant.

Another interesting substrate used in the biogas plant was molasses stillage from ethanol production, also called vinasse. At COD of 333,000 mg/L, the TKN concentration was 19,250 mg/L (**Table 5**), which is quite high. After the meat and bone meal dosing was stopped, the NH4-N concentration in sludge water decreased below 1600 mg/L; however, it increased to almost 4500 mg/L after ca. 200 days of molasses residue loading (between days 500 and 700). Also the volatile fatty acids concentration increased significantly (**Figure 15**) and pH reached values of above eight (**Figure 14**). These changes were not abrupt but gradual and the anaerobic biomass adapted to these new conditions; therefore, the changes had no significant effect on the biogas production. Lower biogas production in this time period was caused by the lack of maize silage, as only one third or one half of the designed amount was loaded. After the molasses stillage loadings were stopped, the volatile fatty acids concentration decreased again (**Figure 15**). In the following season when maize silage was lacking, rye silage or a mixture of oat and peas was used.

Considering the anaerobic reactor operation stability, crude glycerol seems to be a promising co-substrate; it was used for more than two years in the biogas plant. It is a by-product of biofuel production; some of its characteristics are listed in **Table 5**. As it can be seen from the biogas production (**Figure 13**), pH (**Figure 14**), and volatile fatty acids concentration (**Figure 15**), the use of crude glycerol as a co-substrate with maize silage resulted in the anaerobic reactor stabilization.

To evaluate the specific biogas production from crude glycerol and its contribution to the total biogas production, a stable biogas plant operation period of 141 days was chosen, when only maize silage and crude glycerol were loaded to the anaerobic reactor. The average daily biogas production achieved was 4091.4 m3 , at the daily silage loading of 5280.4 kg TS (TS in the silage used was 42.47%) and the daily crude glycerol loading of 683.7 kg. To determine the specific biogas production from crude glycerol, the value of the specific biogas production from maize silage (0.66 m3 per 1 kg of the maize silage TS, obtained when maize silage was the only substrate) was employed. The average daily amount of biogas produced from maize silage was calculated as 3485 m3 and the average daily amount of biogas produced from crude glycerol was 606.4 m3 . The specific biogas production per 1 kg of crude glycerol was 0.887 m3 , which corresponds to the specific biogas production of 0.512 m3 /kg of COD and is in agreement with the results presented in [31].

Biogas produced from crude glycerol represented 14.82% of the total biogas production, while 11.46% of the total TS, 12.1% of VS and only 5.21% of the total mass of the raw materials loaded. At the electrical power output of the cogeneration unit of 300 kW (electrical power output of the cogeneration unit was increased from 276 kW to 300 kW after an agreement with the producer considering the operation experiences), the daily electric energy production from crude glycerol was 1067 kWh and almost 15% of silage were saved.

Amon et al. [32] studied the influence of various loading doses of crude glycerol on the anaerobic digestion of pig manure, maize silage and maize corns. Co-fermentation effect was observed. It means that methane yield of the basic mixture supplemented with glycerol was higher than the combined methane yields of both substrates if digested separately. The cofermentation effect was especially high with glycerin additions of 3–6%. They recommend the glycerol content of maximum 6% for a stable reactor operation.

**Figure 15.** Concentration of VFA in filtrated sludge water from the anaerobic reactor in the biogas plant.

oat and peas was used.

190 Advances in Silage Production and Utilization

reactor stabilization.

production achieved was 4091.4 m3

Another interesting substrate used in the biogas plant was molasses stillage from ethanol production, also called vinasse. At COD of 333,000 mg/L, the TKN concentration was 19,250 mg/L (**Table 5**), which is quite high. After the meat and bone meal dosing was stopped, the NH4-N concentration in sludge water decreased below 1600 mg/L; however, it increased to almost 4500 mg/L after ca. 200 days of molasses residue loading (between days 500 and 700). Also the volatile fatty acids concentration increased significantly (**Figure 15**) and pH reached values of above eight (**Figure 14**). These changes were not abrupt but gradual and the anaerobic biomass adapted to these new conditions; therefore, the changes had no significant effect on the biogas production. Lower biogas production in this time period was caused by the lack of maize silage, as only one third or one half of the designed amount was loaded. After the molasses stillage loadings were stopped, the volatile fatty acids concentration decreased again (**Figure 15**). In the following season when maize silage was lacking, rye silage or a mixture of

Considering the anaerobic reactor operation stability, crude glycerol seems to be a promising co-substrate; it was used for more than two years in the biogas plant. It is a by-product of biofuel production; some of its characteristics are listed in **Table 5**. As it can be seen from the biogas production (**Figure 13**), pH (**Figure 14**), and volatile fatty acids concentration (**Figure 15**), the use of crude glycerol as a co-substrate with maize silage resulted in the anaerobic

To evaluate the specific biogas production from crude glycerol and its contribution to the total biogas production, a stable biogas plant operation period of 141 days was chosen, when only maize silage and crude glycerol were loaded to the anaerobic reactor. The average daily biogas

, at the daily silage loading of 5280.4 kg TS (TS in the silage

To complete the biogas plant monitoring results obtained for various co-substrates, the course of suspended solids concentration in the anaerobic reactor is provided in **Figure 16**, which shows a gradual increase of this concentration in the period of more than two years. After crude glycerol started to be added to the reactor, the concentration of suspended solids slightly decreased, which had a positive effect on the reactor mixing.

In our case of long-term crude glycerol loading in the biogas plant, the average glycerol VS addition was 12.1% and the anaerobic digestion process was stable. However, no co-fermentation effect was observed.

Conclusions of the study of various substrates loading on the biogas plant operation:

Co-substrates used in anaerobic digestion of maize silage as the main substrate can have a positive as well as a negative effect on the biogas plant operation.

Uncontrolled meat and bone meal loading resulted in a failure of the anaerobic reactor due to the high nitrogen content in this substrate. Its processing together with the maize silage is possible; however, the loading dose has to be regulated considering the ammonia nitrogen concentration and the pH in the anaerobic reactor.

It has been proved that the inhibitory effect of ammonia nitrogen depends on also the course of its increase. While an abrupt increase of the NH4-N concentration to approximately 2800 mg/ L (loading of meat and bone meal) resulted in an inhibition of the anaerobic processes, gradual increase to almost 4500 mg/L (in case of vinasse loading) showed no negative effect on the process.

Another alternative is growing energetic crops which are not part of the food chain of humans or animals, for example, sorghum provides interesting hectare yields as well as specific biogas production values [34]. Other such crops include hemp (*Cannabis sativa* L.) [35] or Chinese

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Also biologically degradable waste from agriculture and industry as well as municipal waste are suitable substrates for biogas production and are an alternative to maize silage, which has been confirmed by the estimated biogas potential of these substrates in Germany [37]. While the energetic crops biogas potential was estimated to be 46.2% of the total biogas potential, the rest is obtained from various waste materials such as livestock excrements, harvesting residues and by-products of crops processing, municipal waste, sewage sludge, landscaping and

Although a structure of the substrate mixture used in biogas plants is not sustainable even though its change is inevitable a long-time will pass before maize silage loses its position as

This contribution is the result of the project implementation: Finalizing of the National Centre for Research and Application of Renewable Energy Sources, ITMS 26240120028, supported by

the Research & Development Operational Programme funded by the ERDF.

Faculty of Chemical and Food Technology, Institute of Chemical and Environmental

[1] Landbeck M., Schmidt W. Energy maize-goals, strategies and first breeding successes. CD-ROM computer file. In: Proceedings of the First International Energy Farming Congress, Papenburg, Germany, March 2–4, 2005. Kompetenzzentrum Nachwach-

[2] Amon T., Kryvoruchko V., Amon B., Moitzi G., Buga S., Lyson D.F., Hackl E., Jeremic D., Zollitsch W., Potsche E. Biogas production from the energy crops maize and clover

Engineering, Slovak University of Technology, Bratislava, Slovak Republic

Address all correspondence to: miroslav.hutnan@stuba.sk

sende Rohstoffe, Werlte, Germany, 2005. p. 2–4

silver grass (*Miscanthus sinensis Anderss*) [36].

the main substrate for biogas production.

industrial wastes.

**Acknowledgements**

**Author details**

Miroslav Hutňan

**References**

**Figure 16.** Concentration of undissolved substances in the anaerobic reactor in the biogas plant.

Crude glycerol loading of 12.1% of the total loaded VS had a positive and stabilizing effect on the biogas plant operation.
