Facilitating Biodigester Biogas Production Using a Rumination Model of Bos taurus



In order to develop resource and time efficient methods of biogas production as an energy source, the biodigestion procedure physically and chemically modified the biodigestion process to mimic the rumination process of Bos taurus. The experimental protocol used manure slurry and the independent variables of a varying mixing times and digestive enzymes to produce biogases. The results demonstrated significant increases in biogas production with both mixing time variation and enzyme additives to manure slurry. This research is relevant for applications to improve biogas production of artificial biodigesters and further investigation on the mechanisms for natural production of biogas by Bos taurus.


Anaerobic Digestion Process

The anaerobic digestion process enables biodigestion to produce biogas without oxygen and in the presence of certain types of bacteria (Cavinato, 2011). The four main steps are hydrolysis, acidogenesis, acetogenesis, and methanogenesis:

  1. Hydrolysis

Bacteria break down organic molecule polymers into monomers: Carbohydrates, proteins, and fats to monosaccharides, amino acids, and fatty acids.The primary chemical reaction equation is C6H10O4 + 2H2O ? C6H12O6 + 2H2.

  1. Acidogenesis

In acidogenesis, the hydrolysis products are converted into volatile acids, ketones, alcohols, hydrogen, and carbon dioxide by acidogenic bacteria.

The produced hydrogen, carbon dioxide, and acetic acid skip stage 3 and go directly to stage 4, also known as methanogenesis, because acetogeneic bacteria are not needed to break down these products.

  1. Acetogenesis

Acetogeneic bacteria convert alcohols, propionic acid, and butyric acid into hydrogen, carbon dioxide, and acetic acid. This process is often prolonged in the digestive system of cattle as well as initial biodigestion of artificial biodigesters. The main biogas produced is carbon dioxide.

  1. Methanogenesis

In step 4, methanogens convert hydrogen and acetic acid to methane and carbon dioxide, which results in the larger quantities of methane and carbon dioxide produced by biodigestion.

Biogas Biodigestion

Biodigestion is the biochemical process of fermenting organic material under anaerobic conditions to produce biogas, primarily consisting of methane (“Conservation,” 2015). Methane is produced under these conditions with the addition of water. Bos taurus, or cattle, account for 20% of the fermented production of methane and 7% of manure management methane in the United States alone. Despite the negative environmental effects of methane, including its gaseous ability to capture twenty-three times the amount of heat that carbon dioxide can hold, it can be used for energy production such as for heat and electricity. In addition to methane, natural biogas production also produces carbon dioxide (20-50%), nitrogen (0-10%), hydrogen sulfide (0-3%), and hydrogen (Ishler, 2016).

To produce biogas in a controlled environment, the containers that can be used are called artificial biodigesters, which range from two-liter, homemade bottle systems to industrial buildings and underground vats. There are two common types: batch and continuous biodigester systems. Most homemade systems only add the biodigesting materials (i.e. manure, compost, food waste) one time. Continuous systems are more common on large farms and sewage plants as more biodigesting materials are added over time.

Biogas Codigestion

Codigestion is the process of acquiring biogases by anaerobic digestion with two or more different forms of organic material (Braun and Wellinger, 2007) as opposed to only one type of material, such as manure. This process has been commonly tested with specific carbohydrates, proteins, and lipids (Arsova, 2010), but it has been found that lipids produce the greatest amount of the biogas of the three macromolecules, due to chemical composition. The largest difference in biogas production is about three to four days later for lipids in comparison to digested protein (El-Mashad HM and Zhang R., 2010).


Ruminants are ungulate mammals that chew cud, which is regurgitated from the rumen to physically and chemically break down food into usable nutrients, also generating byproducts that primarily consist of methane and carbon dioxide (Radunz, 2012). The four main internal compartments that undergo rumination are the rumen, reticulum, omasum, and the abomasum.



The surplus of agricultural manure, especially Bos taurus manure, is widely being used to produce biogas as a more environmentally-friendly alternative to non-renewable energy sources. With this in mind, the following experiments’ purpose is to develop resource and time efficient methods of biogas production through modification of the biodigestion process. Investigation of mimicking the natural rumination process of cattle through the addition of digestive enzyme and mixing techniques will be conducted.


Hypothesis 1:

As the mixing time length of arificial biodigesters increases, biogas generation will increase.

Hypothesis 2:

As the amount of amylase enzyme added to artificial biodigesters increases, biogas generation will increase.


Ingesta Breakdown

  1. Physical Breakdown of Ingesta

Physical chewing was modeled in Experiment 1, by mimicking the cud-chewing breakdown of ingesta, which ideally ferments 60-75% of organic material before it is exposed to gastric chemicals and conditions.

  1. Chemical Breakdown of Ingesta

Catabolic enzymes are the catalysts that facilitate chemical digestion (Ziemi?ski and Fr?c, 2012), which was investigated by mixing with manure slurry in Experiment 2. The enzyme chosen was amylase, an enzyme that breaks down carbohydrates. In the mouth, therre are small amounts in saliva, and the pancreas produces enzyme for primary digestive breakdown in the rumen. Another enzyme as a potential contender for this experiment was lipase, an enzyme that breaks down fats (Sissons, 1981). The regulated diets of the Bos taurus consisted primarily of carbohydrates, thus amylase was ideal to test in this experiment.


The independent variables were increments of mixing time lengths tested (0, 15, 30, & 60 minutes) and amounts of amylase tested (0 g, 25 g, 50 g, & 100 g).

The response variable was the mass (g) and rate (g/day) of biogas generated for three data collection dates. After ten, fifteen, and twenty days of biodigestion, the quantitative measurements were taken for comparison.

The consistent factors that were kept the same throughout the conducted experiments included the environmental conditions in testing room (temperature, air flow, lighting, etc), the amount of manure slurry (300 g) used in mixing experiment (Experiment 1) and amylase experiment (Experiment 2), collecting manure slurry from the same batch of manure, the length of time (20 days) allocated for biogas production, as well as the protocol used for measuring biogas production and rate.

A regulated diet was prescribed and fed to the Bos taurus herd that experimental manure slurry was derived from. This diet was specific for the week of January 24th, 2016 to January 31st, 2016 (Manure slurry collected on January 29th, 2016.


The following materials were collected to perform the two experimental procedures:

  • Activated Filter Carbon
  • Amylase Enzyme
  • Apron
  • ATP Vinyl-Flex PVC Plastic Tubing
  • Bos taurus feces
  • Cement Mixer
  • Clamps
  • Cylindrical Bottles (453.6 g)
  • Distilled Water
  • Duct Tape
  • Face Masks
  • Funnel
  • Graduated Cylinder
  • Hot Glue and Glue Gun
  • Laptop
  • Latex Balloons
  • Latex Gloves
  • Markers
  • Metric Electronic Balance Scale (± .01g)
  • Metric Rulers
  • Metric Thermometers
  • Notebook
  • Pencils & Pens
  • pH Test Strips
  • Regulated Diet Mixture
  • Scissors
  • Sodium Hydroxide Solution (1 M)
  • Timer
  • Ziploc® Bags

Experimental Procedures

  1. Mixing Experiment (Experiment 1)

Step 1: Obtained 300 g of Bos taurus fecal slurry.

Step 2: Placed 5 glass balls into the  mixing biodigestion chamber.

Step 3: Repeated Steps 1 and 2 with 19 biodigester systems.

Step 4: Individually mixed 5 biodigesters for labeled as “15 minutes” for 15 minutes in a cement mixer. (Figure 1 demonstrates steps 1-4 as shown below).


Step 5: Repeated Step 4 with 5 other biodigester systems for each other different time increment tested: 0 minutes (Control), 30 minutes, and 60 minutes. Store biodigester systems at 23°C.

Step 6: Repeated mixing each biodigester system every 48 hours for a total of 10 mixing times (20 days).

Step 7: On the 10th day, determined and recorded the amount of biogas produced by each biodigester using the apparatus shown in Figure 2.


Step 8: Repeated Step 2 on the 15th day and 20th day of biodigestion and then calculated the rate of biogas generation (g/day).

Amylase Enzyme Procedure (Experiment 2)

Step 1: Obtained 300 g of Bos taurus fecal slurry.

Step 2: Added 25 g of amylase enzyme into biodigestion chamber.

Step 3: Repeated Steps 1 and 2 with 4 other biodigester systems.

Step 4: Repeated Steps 1 – 3 with 5 other biodigesters for each different amylase amount tested: 0 g (Control), 50 g, & 100 g of amylase enzyme for a total of 20 biodigester systems tested (Figure 3 demonstrates approximate set-up of amylase / manure slurry biodigesters). All biodigester systems were stored at 23°C.


Step 5: On the 10th day, determined and recorded the amount of biogas produced by each biodigester using the apparatus shown in Figure 1 (shown again below).



Step 6: Repeated Step 2 on the 15th day and 20th day of biodigestion and then calculated the rate of biogas generation (g/day).


Experimental Design: Modification of Biodigestion System

After several trials of the experiments, the addition of activated carbon and sodium hydroxide chambers proved to be inefficient in the removal of hydrogen sulfide (Feng et al, 2005) and carbon dioxide. A more controlled biodigestion system that did not have as many compartments was needed to take accurate measurements. Unless these experiments were performed in an air vacuum chamber in a laboratory, the multiple-chamber design would not allow for sufficient airflow through each individual chamber, which was needed to take accurate biogas measurements in the last chamber. The first biodigestion system shown is Design Number 3 (Figure 1), and the final is Design Number 4, and is shown as Figure 4.

screen-shot-2016-12-10-at-2-03-55-pmMixing Time Experiment (Experiment 1): Physical Ruminative Breakdown Simulation

Mixing artificial biodigesters that contained the biodigesting material, or the Bos taurus manure, improved the ability of the biodigesters to produce biogas. All experimental mixing time increments tested significantly produced more biogas in comparison to the control (no mixing) by the twentieth day of biodigestion. Despite the 60 minute group generation the most biogas with the greatest overall generation rate, the 15 minute and 30 minute mixing time increment groups also exemplified increased biogas production. The 60 minute mixing group of biodigesters averaged the greatest gas production peak of all mixing groups with 4.04 g of biogas produced per biodigester and a biogas production rate of 0.21 g/day. The control group averaged to produce 0.67 g of biogas over the twenty days of the experiment. All biogas collection dates provided statistically significant data groups according to the one-way analyses of variance (ANOVA) and post-hoc tests performed on each individual day of data collection (10, 15, and 20 days). The results are validated by lower p-values than the significance interval of 0.05, which rejects the null hypothesis for each data collection date. After analyzing this data and performing statistical analyses, the results from the experiment show that biogas production was greater for the longer mixing times.

screen-shot-2016-12-10-at-2-05-27-pmAmylase Enzyme Experiment (Experiment 2): Chemical Ruminative Breakdown Simulation

The chemically digestive simulation of the Bos taurus rumination system using amylase enzyme as an additive to biodigest manure slurry significantly increased biogas production. Significant differences between the control and experimental groups tested were observed and proven with one-way ANOVA statistical analyses. The p-values calculated were lower than the set significance interval of 0.05, and therefore rejected the null hypothesis for each data collection date (10, 15, and 20 days). The group with 100 g of amylase added averaged 4.55 g of biogas produced per biodigester and 0.25 g produced per day. The results show that as more amylase was added, more biogas was produced over the twenty day testing period.

screen-shot-2016-12-10-at-2-06-25-pmPost-Experimental Analysis

To further validate the biogas production and rate of the artificial biodigesters tested, as well as investigate the amount of mass that was converted to biogas, the biodigesters were transferred to a laboratory to be analyzed for determination of dry biomass and ash of manure slurry tested. These measurements provide insight of each experimental (independent) variable’s efficiency of breaking down the biodigesting material for anaerobic digestion. The dry mass percentages, dry biomass, dry mass fraction averages of triplicates, and the standard deviation of the triplicates of the mixing time experiment were determined The amylase enzyme experimental contents were not tested for dry mass content due to the different independent variable that would skew the results without the adequate initial experimentation measures previously taken to determine the correct dry mass percentages and dry biomass of the samples. The dry mass percentages were not entirely supportive, but the 60 minute mixing group did show lower percentages of dry mass overall.

Furthermore, the initial concentration of biogas produced in the artificial biodigesters measured on the 10th day of biogas production demonstrated an overall greater mass of biogas produced (g) per biodigester in comparison to the amounts of biogas produced on the 15th and 20th days. This was determined according to the cumulative rate (g/day) of biogas produced. Most experimental groups tested demonstrated a decline in the rate of biogas being produced, except for the experimental groups that received the highest intensities of the independent variable being tested (i.e. the 60 minute mixing group, 50 g amylase group, and the 100 g amylase group). Most likely, the biogas being produced in the latter time frame of the experiments was mainly methane instead of carbon dioxide, due to the reasons stated in the Discussion. Methane also has a lower density than carbon dioxide, which would not produce as great a mass of carbon dioxide per mole, which can also provide an explanation as to why the mass-based rate of biogas produced ultimately decreased over time.

In a solid biofuels research laboratory with the samples from this study, the biodigested material was analyzed for dry mass percentages to determine the efficiency of the biogas production, justifying or contrasting the concluded results. In general, the 60 minute mixing group demonstrated lower percentages of dry mass. The lower percentages indicate more biodigested material converted into biogas during the experiment. The dry biomass (g) measurements, according to the dry mass percentages, also demonstrate scatter plot-like trends from the control to the 60 minute mixing group tested.


Before taking quantitative data, visible differences could be seen with the amylase groups of Experiment 2. The biodigestion chamber was so full of air that the bottle was expanding. Evidently, the 100 g amylase additive group produced the most biogas with the greatest rate out of all three experiments. The enzyme acted as a catalyst to break down carbohydrates and facilitate anaerobic digestion. Amylase was most efficient when biodigested with manure, and was relatively cost-efficient, as well.

The mixing groups produced sufficient results as well, with no additives. In that case, mixing would be the most cost and resource-efficient method in an industry setting to produce large amounts of biogas in relatively shorter time frames, when comparing to the control group results. The mixing of the biodigestion materials simulates the physical chewing of organic materials by Bos taurus, and the physical breakdown of the material is possibly the reason why more biogas was produced for the longer mixing time increments.

Several nuisance variables could have affected the experiments without human or mechanical ability to control these factors. Some of these variables could include slight differences in moisture content of the material in the artificial biodigesters, minor leaks of biogas in biodigesters during testing, differences in amount of waste products produced by the acidogenic and acetogenic bacteria on biogas production

One major question that still remains unanswered is the exact amounts of biogases being produced in the biodigesters. Some scientific assumptions can be made regarding the biogases produced based off of research of the anaerobic process and chemical compound properties. Due to the relatively fresh manure slurry content of the manure, the cows had been producing more carbon dioxide than methane (only about 20%) in the rumination process, because the process is relatively quick (2 to 3 days). This is because of a main acid being produced during the 2nd stage of the anaerobic digestion process: carboxylic acid. In acidogenesis, the carboxylic acid is converted to produce carbon dioxide. Acetogenesis, the 3rd stage, produces acetic acid, which is the main component that involves methanogenesis and the production of methane. Traditionally, artificial biodigesters run similarly to a train in the sense that it will start off slow, or one way, and end up gaining momentum and speed after a certain time, which is different from the initial start. The carboxylic acid is already mainly being produced in Bos taurus, and this is predicted to carry over to the biodigesters if the slurry is fresh, which was the circumstance in this experiment. Therefore, more carbon dioxide would most likely be produced. In time, the biological and chemical materials in the biodigester can produce more methane with the production of acetic acid, from the 3rd stage of anaerobic digestion, or acetogenesis. The carboxylic acid is not being as prevalently produced because the anaerobic bacteria are converting more acetate to be used in the 4th stage of anaerobic digestion instead of using the produced carboxylic acid for carbon dioxide production. After the volatile fatty acids are synthesized, there is enough of the substance to convert into larger amounts of acetate, which is required for methanogens to produce methane. Therefore, if carboxylic acid is being produced initially from previous anaerobic digestion (Rumination of the Bos taurus), then it will take time to switch from producing the main gaseous byproduct of the 2nd stage (Acidogenesis), which is carbon dioxide, to methane, which is the main gas produced in the 4th stage (Methanogenesis). To justify the greater likeliness of carbon dioxide production during the initial portion of the experiments, the possible masses (g) of carbon dioxide, methane, and air for several measured volumes (cm3) of the biogas collection balloons from the 10th day of biodigestion were calculated in 100%, 66.6%, and 33.3% mass possibilities. The theoretical calculations do not completely correspond with the measured masses (g) of biogas produced and the measured volumes (cm3), but the carbon dioxide masses (g) calculated for the volumes (cm3) were nearest in value to the measured biogas masses (g) in comparison to the methane and air (Pidwirny and Jones, 2014) masses (g) calculated (Trautman and Richard, 1996). One example table demonstrating the greater theoretical masses (g) of carbon dioxide for the 15 minute mixing group (Biodigesters 1-5) for the 10th day of biodigestion data collected is shown below in Table 1:


Along with the likelihood of the initial biogas produced being carbon dioxide, the theoretical measurements of a film of condensed water vapor are likely possible in addition to the carbon dioxide masses calculated (g). According to Project E24744: Film Condensation in a Horizontal Rectangular Duct (Lu and Suryanarayana, 1992) performed at Michigan Technological University for NASA research, water condensate films within certain containers typically ranges between 1.0 mm to 3.0 mm in thickness . The latex balloons for the same volumes (cm3) originally measured was calculated to determine the water condensate film thickness range of 0.9 mm to 0.23 mm for all balloons. This was determined after the area (cm2) and residual volumes (cm3) of the balloon contents were calculated. One table representing the 15 minute mixing group data for these theoretical calculations with 100% carbon dioxide (initial biogas) estimates is produced below in Table 2.


The most feasible explanation of the masses (g) calculated representing biogas production was most likely initially a mixture of water condensate film and carbon dioxide, according to the theoretical calculations (performed for all biodigestion data collected on 10th day with only 15 minute mixing data shown).


Mixing Time Experiment Conclusion

With the data obtained from Experiment 1, there is enough evidence to conclude that increased mixing time increments produced greater amounts of biogas over the twenty-day biodigestion period. On average, the 60 minute mixing group of artificial biodigesters produced the greatest amount of biogas out of all of the experimental groups tested.

Amylase Enzyme Experiment Conclusion

The data of Experiment 2 supports Hypothesis 2 because biogas production was greater per biodigester as the amount of amylase added to the artificial biodigester increased. The experimental group with one hundred grams of amylase added had the highest overall averaged amount of biogas production as well as the fastest generation rate.  

Improving Biodigestion Protocol

Future Improvements and Changes

With more trials (3 – 10 trials) and testing a greater number of artificial biodigesters (? 30), the results could be analyzed more accurately with the larger sets of data and trials. By testing a greater range of different amounts of amylase, testing a larger-scaled biodigester containing greater amounts of biodigestion materials, and/or using a different protocol for measuring the amount and rate of biogas being generated could possibly improve results in the future. Another addition to improve this experiment could be to prolong the time allotted for biodigestion, such as 30 or 40 days instead of the 20 days specified in the procedures.

Possible Future Experiments and Extensions

Some future experiments or extensions that could be investigated based on the results of these experiments could include a variety of topics. One example is to create a solar-powered mixing apparatus to add to biodigesters to test for continuous mixing. An experiment to determine the effects of different diets of Bos taurus on biogas production could have application for farmers with biodigesters. A biodigester that separates the biogases produced could be designed.This experiment attempted to improve the slow biogas production issue that prevents biogas from being a more dominant energy source. After concluding that mixing biodigesters and amylase additives facilitate biogas production, more research can be performed to produce more biogas in shorter time spans.

Real-Life Applications

After performing this research, the main issue to consider is the effectiveness and usability of these methods of artificial biodigestion to be applied for energy production. A major environmental concern of today is the usage of nonrenewable energy, and the efforts to primarily turn over to renewable energy sources to protect ecosystems. The primary issue of replacing natural gas with biogas is the cost factor: natural gas is on average $3.86 per one million British Thermal Units (MMBTUs) versus $10.00 to $13.00 per MMBTU of biogas (“Biogas and Biomethane,” 2016). With this research, the results show that more biogas was produced within the experimental time frame by mixing artificial biodigesters and adding the amylase enzyme to the slurry in comparison to the control artificial biodigesters. The means that the price of biogas could potentially be lowered if more biogas was produced in shorter time frames by implementing new methods such as the mixing or amylase additive procedures to artificial, mass energy-producing biodigestion systems.


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This experiment would not be possible without the guidance of Mr. Donald Orlowski of Freeport Area Middle School, a science teacher that has a true passion for what he does. Two other individuals from Pennsylvania State University: Dr. Thomas Richard and Ms. Kay DiMarco for their help and advice with these experiments and the laboratory analysis. The Arner Dairy Farm supplied the Bos taurus and other supplies needed to obtain the main materials for this experiment, as well as the facilities to perform the research.


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