The effects of crude glycerol addition on biogas production

Crude glycerol is a by-product of biodiesel production in which the number increases every year and is quite an expensive purification process to meet the technical standards required by consumer industries. To overcome this, it is important to find alternatives for crude glycerol utilization to increase the economic feasibility of the biodiesel industry. This study aimed to evaluate the technical feasibility of biogas production with the addition of crude glycerol from a Palm Oil Plant. The glycerol was introduced into 500mL Anaerobic Digester (AD) as a carbon source and energy source for the growth of methanogenic bacteria along with cow dung and Distilled Water with a ratio of 1:1. The addition of crude glycerol was 5% wt (GL5), 10% wt (GL10) dan 15% wt (GL15), and one control reactor without crude glycerol addition (GL0). AD was operated in a batch system at mesophilic conditions for 30 days. The highest biogas yield was obtained in the experimental set GL10 as much as 380 mL/g VS and was formed on the 3rd day but the highest percentage of methane gas (CH4) was obtained from the control set GL0 as much as 60.2%. In addition to the identification of bacteria, it was found that the type of Bacillus sp in the GL10 treatment was the most biogas producer, and based on the results of its bio-slurry analysis it could be used as organic fertilizer and soil improvement for agriculture and degraded soil.


Introduction
Some countries use biodiesel in the primary energy mix to reduce dependence on fossil fuels and to promote sustainable development.Biodiesel production in Indonesia has increased significantly from 190,000 kilolitres in 2009 to about 8.4 million kilolitres in 2019 according to data from the Indonesian Ministry of Energy and Mineral Resources.Biodiesel is commonly produced by using the transesterification process plant where oil is reacted with an alcohol, such as methanol, and aided by a catalyst, such as potassium hydroxide (KOH).The resulting product is fatty acid methyl esters (FAME), which is popularly called biodiesel, as well as by-products i.e., crude glycerol that has a relatively low degree of purity.The biodiesel formation process is shown in Figure 1.
For every kilogram of biodiesel produced, about 100 g of glycerol is produced as a by-product of the transesterification reaction [1].Glycerol (or glycerine or 1,2,3-propanetriol) with the chemical formula C3H5(OH)3 is colorless, odorless, viscous, and non-toxic alcohol that melts at 17.8 o C [2].Crude glycerol is produced by homogeneous transesterification alkaline catalyst contains about 50-60% of glycerol, 12-16% alkali particularly in the form of soap and alkali hydroxide, 15-18% methyl ester, 8-12% methanol, 2-3% water, calcium, magnesium, phosphorus or sulfur, derived from primary oil and sodium or potassium from catalysts [3,4] Figure 1.Biodiesel Formation Process (Source: [5]) To maintain the economic and environmental sustainability of biodiesel production, it is important to look for alternative use of crude glycerol.This is due to trade at a competitive price, crude glycerol must undergo an expensive purification process to meet the technical standards required by industrial consumers.One possible solution is the recovery of crude glycerol as carbon and an energy source for the growth of industrial microbiological microbes [5] by producing biogas in a biodigester [6].This biogas is a gas produced by anaerobic activity or anaerobic fermentation of organic material as shown in Figure 2. Biogas mostly contains methane (CH4), carbon dioxide (CO2), and some small number of gases (H2, H2S, NH3).The energy contained in biogas depends on the methane concentration (CH4).The higher the methane content, the greater the energy content (calorific value) of the biogas and vice versa.Production of biogas through anaerobic digestion offers significant advantages compared to other forms of processing of crude glycerol.It requires lower investment and more modest operating conditions compared with more sophisticated pre-processing technology.In a process of biogas forming in a digester that uses bacteria as a means to break down polymer compounds (in this case carbohydrates, fats, and proteins) additional organic material is needed to help speed up the process.Numerous studies have been conducted, [8] added 6% of crude glycerol into pig manure and corn silage and amount of biogas production to 1.114 and 0.679 m 3 /kg respectively, and increased the proportion of methane 5.6% and 9.8% each.[9] also compared biogas production in two reactor batches, the first with a mixture of water and cow dung (1:1) and the other using the same mixture but with 5% (v/v) glycerol supplementation, which is equivalent to 0.221 kg of glycerol.The researchers reported that after 14 days of reaction time, the reactor containing glycerol can increase the production of methane by around 4L.In this study, several Anaerobic Digesters (AD) will be made with a higher composition of crude glycerol, namely 5%, 10%, and 15%, and coupled with cow dung as a natural starter without special treatment of factors that can increase biogas production.

Biodigester in Laboratory
Four Anaerobic Digester (AD) systems were made on the laboratory scale, using 500mL volume filtering flasks.The upper side of the AD was closed with a rubber plug and a hole was made that was connected with a 10-inch plastic hose to a 100mL plastic syringe while on the other side, the plastic hose was connected to a 12mL plastic syringe.The syringes were used to measure the biogas volume that was formed.In this study, no additional treatments for AD and only used at room temperature or environmental conditions.Stirring AD was only done once after all feedstock was put in AD and its pH was measured.Before closing, the AD that contained the feedstocks was purged by using nitrogen gas for 60 seconds to remove oxygen-containing air from inside the AD.Biogas monitoring was done daily for 30 days.The AD installation used in this research can be seen in Figure 3.

Materials
Cow dung was collected from cattle farms owned by residents in Koto Lua sub-district, Padang City, West Sumatra.The cow dung used for this experiment was a fresh one.In its use, cow dung was homogenized using distilled water with a ratio of 1:1.Crude Glycerol used is a by-product from a Biodiesel Production Plant in Dumai City, Riau Province.There was no dilution treatment for the crude glycerol.Preparation of bacterial growth medium Tryptic Soy Jelly (TSJ) conducted in Microbiology Laboratory of Environmental Engineering Department, Universitas Andalas.

Experiment Setup
Four treatments were tested in experiments by setting the ratio between cow dung and distilled water 1: 1.The first control set (GL0) without the addition of crude glycerol, then the experimental set GL5, GL10, and GL15, with the proportion of adding 5%, 10%, and 15% weight, each of the distilled water was replaced with crude glycerol as shown in Table 1.The experiment was carried out in a batch process and a mesophilic environment (temperature 26-29˚C).Observations on the formation of biogas were carried out for 30 days.

Method of Analysis
The biogas results were recorded every day by observing the movement of the plastic syringe which showed the amount of biogas produced.The pH of feedstocks was first measured at baseline in AD and re-measured after 30 days.The ambient temperature was recorded every day using an indoor ambient thermometer PCE-FWS-20.The proportion of methane gas itself was measured by using a Gas Analyzer 5000 as shown in Figure 4.While the identification of microbes that produced the highest biogas was analyzed at the Veterinary Institute Bukittinggi (BVET) with a single colony morphology observed visually as edge shape, the color of the colony, the state of the colony, and the nature of gram with 3% KOH test, moreover biochemical tests of bacteria were also carried out.

Feedstock Analysis and Experimental Sets for Anaerobic Co-Digestion
The raw materials for this research were cow dung, crude glycerol, and distilled water.Cow dung is used as an inoculum for Anaerobic Digester (AD).pH, moisture, Total Solid (TS), C/N ratio data for raw materials is shown in Table 2. Table 2 shows that the C/N from cow dung was 18.32 while crude glycerol was 12.84.This condition will affect the formation of biogas, where the ideal C/N is around 25-30 [10].The increase in carbon content will give rise to more carbon dioxide formation and lower pH value, while the high value of nitrogen will enhance the production of ammonia gas that could increase the pH to the detriment of the micro-organisms [11].

Biogas Production during Co-Digestion
In general, the anaerobic process is an activity of breaking down organic materials by microorganisms in the absence of oxygen.Decomposition of organic compounds such as carbohydrates, fats, and proteins contained in the liquid waste with anaerobic processes will produce biogas containing CH4 gas percentage (50-70%), CO2 (25-45%), and a small amount of nitrogen (N2), hydrogen (H2 ) and hydrogen sulfide (H2S).In the AD control set, it appears that the result of biogas accumulation for 30 days was 120 mL g VS.Whereas the highest biogas yield (235 mL/g and 380 mL/g, respectively) was produced by the GL5 and GL10 set experiments, while the addition of crude glycerol by 15% did not produce biogas as shown in Figure 5.The addition of excessive crude glycerol to the sample will cause a rapid change in pH value which takes a long time for methanogens to adjust, overcoming the inhibition of methane production [12].The best thing to do is to add crude glycerol gradually to the continued system to allow the bacteria to adjust to their environment.The microorganisms involved in the Anaerobic Digester (AD) differ greatly in terms of physiology, nutritional requirements, growth kinetics, and sensitivity to environmental conditions.Failure to maintain a balance between groups of microorganisms is the main cause of reactor instability [13].This inhibition is usually indicated by a steady-state rate reduction of methane gas production and accumulation of organic acids [14].Based on Figure 5 several factors affect the formation of biogas: 1. Organic material (substrate).
The concentration of substrate may affect the work processes of microorganisms.The optimum conditions can be obtained when the number of microorganisms is proportional to the substrate concentration.

C/N Ratio
Carbon (C) is used as a source of energy and Nitrogen (N) is needed by microorganisms as a source of nutrition for the formation of body cells.The low C / N value can cause ammonification and poison the bacteria in the digester [15].Ammonia nitrogen in high concentrations or above 3000 mg / L can inhibit the anaerobic fermentation process and will be toxic at any pH.

Acidity (pH)
pH affects the growth and activity of microorganisms.Methane organisms are very sensitive to pH changes.By the time the mixture has become well buffered, which inserted acid/base in large amounts, the mixture will be stable by itself at pH 7.5-8.5 [16].When the mixture has been steady, it is possible to add a small amount of material regularly and can maintain a constant production of gas and sludge (on a continuous flow digester).When the material is inserted irregularly (batchtype digester), the enzymes will accumulate so that organic solids become bad and stop methane production [16].Figure 6 shows the difference in pH before and after the Digestion process in AD.  .Toxic Among the impurities that crude glycerol derived from biodiesel, these three impurities may significantly affect the metabolism of microbes because they are toxic, Long Chain Fatty Acids (LCFA) [17], chloride (Cl) [18], and potassium and sodium salts.LCFA is derived from triglycerides in a transesterification reaction and dissolved in glycerol [3].The results of LCFA fat degradation have the potential to inhibit methanogenesis [19].LCFA which is absorbed by bacteria causes flotation and cell precipitation.This creates a physical barrier that inhibits the transfer of substrates and products, thereby causing inhibition in methane gas production [20].Chloride (Cl -) in crude glycerol comes from hydrochloric acid which is used in the neutralization step of the transesterification process.The chloride concentration causes strong inhibition of methanogenesis usually in the range of 4-9 g Cl − /L [21], and glycerol derived from biodiesel can contain between 34 -46 g Cl − /L.Crude glycerol will be toxic to the anaerobic consortium when used as a pure substrate solution or undiluted.The toxic effect of chloride on microorganisms occurs through cell plasmolysis, namely shrinkage of cell volume by loss of water or loss of cellular activity due to high osmotic pressure [21].Anaerobic microorganism metabolism is also negatively affected by the high salinity of the crude glycerol phase [22].Na or K salt content is relatively high and is derived from the catalyst used for the production of biodiesel.The optimum concentration of sodium ranges from 100-200 ppm and the inhibitory limit is very strong 8000 ppm, while for potassium the optimum concentration ranges from 200-400 ppm and the limit of the inhibitor is very strong around 12000 ppm [12].6. Organic Loading Rate (ORL) ORL is the weight of organic matter per day applied to the specific volume of the reactor.The parameter is COD (Chemical Oxygen Demand) which indirectly represents the number of organic compounds in the sample.It is used to measure the oxygen needed to degrade organic matter.The decrease in specific methane production occurs when the ORL is increased further [5].

Mixing/Stirring
Stirring aims to homogenize the substrate with microbes.Stirring is very beneficial for materials that are in AD because it provides the opportunity for the material to remain mixed with bacteria and allows all materials to undergo an anaerobic fermentation process evenly.For the substrate that is not stirred, it can inhibit the release of biogas due to the formation of foam in the reactor [23].

Starter
A starter containing methane bacteria is needed to accelerate the process of anaerobic fermentation.
The natural starter can be in the form of sludge, organic waste heap, sewage water, or septic tank liquid.Semi-artificial starters, namely from the biodigester facility in an active stage, and artificial starters, namely bacteria that are cultured in a laboratory with artificial media.9. Hydraulic Retention Time (HRT) HRT is the number of days of the digestion process in the AD tank starting from the entry of organic matter until the initial process of biogas formation in the anaerobic digestion process.The length of HRT is highly dependent on the type of organic material (feedstock substrate) before the digestion process is carried out.Cow dung takes 20 ̶ 30 days and part of the gas is produced in the first 10 to 20 days [16].In this study, GL5 and GL10 began to produce gas on day 3.

Methane Gas Production (CH4) During Co-Digestion
As shown in Figure 8, the methane gas production control set is much higher than the experimental set.The total accumulated methane production for the experimental set GL5, GL10, and GL15 were 14.9%, 3.5%, and 0%, lower respectively than the control set which produced 60.2% methane gas.The more crude glycerol was added, the less the total accumulated methane production is obtained.AD GL15 has a toxic condition due to the addition of excessive crude glycerol.

Figure 8. The Concentration of Methane Gas and Crude Glycerol Composition
Biogas produced from the fermentation process of organic waste does not contain 100% combustible gas because the biogas product consists of methane (CH4), carbon dioxide (CO2), nitrogen (N2), hydrogen (H2), hydrogen sulfide (H2S), oxygen.(O2) and water content (H2O) (Burke, 2001).The elements that play a role in determining the quality of biogas are methane gas (CH4) and carbon dioxide (CO2).When looking at the volume of biogas formed, GL10 produces the highest volume of biogas but produces a lower methane concentration than the control set (GL0) or low methane purity.The purity of methane from the biogas product is important because it will affect the resulting calorific value.If the CH4 level is high, the biogas will have a high heating value.Conversely, if the CO2 level is high, it will result in a low calorific value of the biogas [24].The quality of this biogas can be improved by treating

Analysis of Methane (CH4) Forming Bacteria
One factor that is crucial in the process of the formation of biogas is the role of bacteria because the conversion of organic matter into biogas is the result of a variety of bacteria that work in the consortium [25].Based on the analysis of biogas formation, the GL10 experiment set produced the most biogas, namely 380 mL/g VS which was formed from bacterial activity.Microorganisms in large numbers can grow on media containing glycerol and use it as a source of carbon and energy.Identifying bacteria, it can be done by observing the microscopic-macroscopic characteristics and biochemical tests of bacteria.
For bacterial identification preparations as shown in Figure 9 (A).There are four groups of bacteria involved in the process of biogas formation, namely hydrolytic bacteria, acetogenic bacteria, homoacetogenic bacteria, and metanogenic bacteria.For methane gas itself, four types of anaerobic bacteria play a role in producing methane gas, namely Methanobacterium, Methanobacillus, Methanococcus, and Methanosarcina.Based on this study, the bacteria present on the set of AD Experiments GL10 can be seen in Figure 9 (B) and has the characteristics shown in Table 3.Based on the results of bacterial identification, these bacteria have Kingdom: Procaryotae; Phylum: Bacteria; Class: Schizomycetes; Order: Eubacteriales; Family: Bacillaceae; Type: Bacillus sp.[27].This is research [28], that Bacillus sp. is a gram-positive bacterium, white, flagellated, not slimy, and has a jagged edge shape.

Analysis of Potential Liquid Fertilizer from Bio-Slurry of Anaerobic Digester
Bio-Slurry is waste generated from Biogas Anaerobic Digester (AD), formed in liquid and solid which is very useful as a source of nutrients for plants [29].In this study, AD GL10 bio-slurry made from cow dung and crude glycerol was analyzed.The results of the analysis of the initial characteristics of bioslurry raw materials can be seen in Based on Table 4, it can be seen that the pH and C-Organic values meet the standards while the nutritional values of N, P, and K have not met the standard values.To get good nutritional content in liquid organic fertilizers, it is necessary to add supporting raw materials that can add nutritional value, especially the value of N, P, and K.This waste can be used as fertilizer and soil improvement for agriculture and degraded soil.

Conclusion
This study demonstrated that the crude glycerol from the biodiesel production process can be used as a raw material to increase the production of methane during the process of co-digestion with cow dung.Crude glycerol from biodiesel production can be a suitable carbon source for biogas production under mesophilic conditions (26-29˚C).The addition of cow dung causes an increment of biogas production at a faster retention period but a lower methane content than the biogas production from cow dung alone.This of course requires gas purification to obtain higher methane gas (CH4).This study leads to the conclusion that the optimal proportion of crude glycerol added as a supplement to cow dung in the Anaerobic Digester (AD) is between 5% -10% and the addition of crude glycerol of more than 10% can cause conditions in AD to become toxic which causes microorganisms to not developed.This research also shows that the recovery of crude glycerol from biodiesel production can transform aerobic treatment and biogas production into more attractive options, thereby contributing to saving greenhouse gas emissions and being more efficient for producing renewable energy.

Figure 6 .
Figure 6.Changes in pH on Digestion Anaerobic Digester (AD) 4. Temperature Temperature greatly determines the length of the digestion process in the Anaerobic Digester (AD).When the temperature increases, generally biogas production also increases following the limits of the ability of bacteria to digest organic waste.Generally, small-scale AD works at mesophilic bacteria temperatures with temperatures between 25-37℃.In this study, the AD temperature was

Figure 7 .
Figure 7. Anaerobic Digester (AD) Environmental Temperature5.ToxicAmong the impurities that crude glycerol derived from biodiesel, these three impurities may significantly affect the metabolism of microbes because they are toxic, Long Chain Fatty Acids (LCFA)[17], chloride (Cl)[18], and potassium and sodium salts.LCFA is derived from triglycerides in a transesterification reaction and dissolved in glycerol[3].The results of LCFA fat degradation have the potential to inhibit methanogenesis[19].LCFA which is absorbed by bacteria causes flotation and cell precipitation.This creates a physical barrier that inhibits the transfer of substrates and products, thereby causing inhibition in methane gas production[20].Chloride (Cl -) in crude glycerol comes from hydrochloric acid which is used in the neutralization step of the transesterification process.The chloride concentration causes strong inhibition of methanogenesis usually in the range of 4-9 g Cl − /L[21], and glycerol derived from biodiesel can contain between 34 -46 g Cl − /L.Crude glycerol will be toxic to the anaerobic consortium when used as a pure substrate solution or undiluted.The toxic effect of chloride on microorganisms occurs through cell plasmolysis, namely shrinkage of cell volume by loss of water or loss of cellular activity due to high osmotic pressure[21].Anaerobic microorganism metabolism is also negatively affected by the high salinity of the crude glycerol phase[22].Na or K salt content is relatively high and is derived from the catalyst used for the production of biodiesel.The optimum concentration of sodium ranges from 100-200 ppm and the inhibitory limit is very strong 8000 ppm, while for potassium the optimum concentration ranges from 200-400 ppm and the limit of the inhibitor is very strong around 12000 ppm[12].6. Organic Loading Rate (ORL)ORL is the weight of organic matter per day applied to the specific volume of the reactor.The parameter is COD (Chemical Oxygen Demand) which indirectly represents the number of organic compounds in the sample.It is used to measure the oxygen needed to degrade organic matter.The decrease in specific methane production occurs when the ORL is increased further[5].7.Mixing/StirringStirring aims to homogenize the substrate with microbes.Stirring is very beneficial for materials that are in AD because it provides the opportunity for the material to remain mixed with bacteria

Figure 9 (
Figure 9 (A) Observation of GL10 Set Experimental Sample Preparations in NA Medium.

Table 1 .
Raw Material Content in Anaerobic Digester (AD)

Table 2 .
Feedstock Characteristics for Anaerobic Co-Digestion

Table 3 .
Identification of Bacteria in Anaerobic Table 4 and compared with the Regulation of the Minister of Agriculture Republic of Indonesia Number 70/Permentan/SR.140/10/2011concerning Organic Fertilizers, Biological Fertilizers, and Soil Improvement.