Paper No. 984102

EFFECTIVENESS ADDITIVES TO INCREASE ORGANIC   CONVERSION IN ANAEROBIC DIGESTION

by

 Written for presentation at the 1998 ASAE Annual International Meeting sponsored by ASAE Disney's Coronado Springs Resort   Orlando, Florida July 12-16, 1998

 Summary:

Bench scale tests of twelve 3-L working volume anaerobic digesters receiving various commercial additives designed to enhance the fermentation process were completed using primary municipal sludge as the organic material. Two additives showed positive results, two showed essentially no change, while one showed a negative result to the fermentation process. Using one of the additives showing a positive effect, a level one cost analysis was completed resulting in a benefit to cost ratio of 1.9 when the additive is included. Increased total volatile solids reduction of 8.2% was realized (P<0.05) for the best additive when compared to the control.

 Keywords: Enzymes, Wastewater, Municipal

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ABSTRACT

 A bench scale analysis of twelve 4-liter anaerobic digesters with a 3- working volume was operated to examine the effects of five commercial additives on the degradation of primary municipal sludge. The reactors were loaded with 150 ml per day of 3% volatile-solid content waste collected from the Lubbock Municipal Treatment Plant. Ten of the reactors were loaded with one of five commercial additives, in duplicate, with two reactors serving as the control. The objective of the experiments was to determine the effects that the additives would have on the performance of the digestion of the waste and the microbial populations. It was found that during steady state operation, the total volatile solids content of reactors receiving the Biocope additive were significantly lower (16.5%) than the control (P<0.05), for an overall increase in volatile solids degradation of 8.2%. A preliminary level- one economic feasibility test showed that $193,257 could be saved over an assumed 20 years of operation, by constructing an 8.2% down-sized treatment plant with the inclusion of Biocope based on an average flow rate of 1 million gallons per day of wastewater into the treatment plant. This provided a benefit to cost ratio of 1.9, which is quite substantial for a wastewater treatment facility.

 The HMB-IV device used to measure aerobic activity of a sample was shown to be effective in determining unsteady mixing in the anaerobic digesters and early detection of a drop in pH below the threshold value of 6.5. HMB-IV readings significantly dropped 1-2 weeks before a pH drop would be detected that would inhibit the digestion process. It was concluded that the HMB-IV device could be excellent for early detection of process failure thereby providing an early response to corrective action.

 INTRODUCTION

 Wastewater treatment is of concern for every city in the world. If waste goes untreated, it can lead to offensive odors and serve as a breeding ground for pathogenic organisms. Anaerobic digestion is one of the oldest and most widely used processes for the stabilization of sludge, due to the fact that it decreases the solid content and reduces the pathogenic organisms without the need for added oxygen. Current anaerobic technology is only capable of partially treating waste in a conventional wastewater treatment system. High levels of degradation require longer retention times or further treatment by aerobic treatment methods, which add to the cost of treatment. Since improvements in biological remediation techniques are necessary, enzyme technology is receiving increased attention. Key microorganisms and enzymes can be isolated to assist in a more complete breakdown of waste under anaerobic conditions. The primary objective of this research was to determine the technical feasibility of using commercially prepared microorganism or enzyme mixtures to enhance the degradation of wastewater at a municipal wastewater treatment system. Secondary objectives were to determine the change in the populations of organisms within the system and to determine the economic feasibility of using these commercial products in a wastewater treatment system. If inclusion of these biological catalysts increases the rate at which waste is broken down, it could lead to the design of smaller treatment facilities, and consequently a reduced cost in the treatment of wastewater.

 There has been increasing interest in the use of biological processes to catalyze reactions. Microorganisms secrete enzymes to break down potential food sources. These enzymes are proteins that consist of chains of amino acids. Enzymes act as a catalyst in breaking down substrates or joining substrates into a larger molecule that is readily available to the microorganisms. Each enzyme is highly specific and usually will only catalyze one particular reaction. Technology exists to isolate enzymes from the microorganisms that created. them, allowing the enzyme to degrade waste without the need for the microorganism. A biocatalyst can refer to a consortia of microorganisms, a particular organism or enzyme, or a mixture of organisms and enzymes that can facilitate a given reaction, since the enzymes created are the catalyst being used (Tramper, 1996).

 Enzymes were first proposed for the treatment of waste in the 1930's, but it was not until the 1970's that enzymes were used to target specific target pollutants in waste (Aitken, 1993). These biocatalysts can be effective in some applications and useless in others. Biocatalysts are often selected over other chemical conversion methods when the number of reaction steps is greatly reduced, faster reaction rates are achieved, or a higher yield of a particular product is achieved without a tremendous increase in cost. Another question to consider when selecting a biocatalyst is when to use isolated enzymes, microorganisms, or a mixture (Tramper, 1996). Aitken (1993) reports several instances where biocatalytic treatment may befavorable. The first is for the removal of a specific chemical from a waste stream before conventional biological treatment methods are applied. The biocatalyst may be able to alter an otherwise toxic or inhibitory compound into something that is acceptable to the organisms used in treatment. A second instance where the use of only enzymes may be favorable is with dilute wastes that are not treatable by conventional biological means like ground water pump-and-treat systems. Other uses include the polishing of a treated wastewater for disposal into the environment and in-plant treatment at the source of generation for possible reuse of the water for other applications.

Use with Insecticides 

One field that has researched biocatalyst use is in the treatment of insecticides. It was found that a mixed bacterial culture growing on the insecticide parathion could hydrolyze the parathion 2,450 times faster than conventional chemical hydrolysis (Munnecke, 1975). In one experiment, this same biocatalyst was used to hydrolyze eight different organophosphates, and the results showed that seven of the insecticides were hydrolyzed at rates between 40 and 1005 times faster than chemical hydrolysis (Munnecke, 1975). In another study, Copella et al., (1990) used genetic engineering to isolate the enzyme parathion hydrolase for in in-situ treatment of organophosphate. Although some strains of microorganisms and genetically altered microorganisms degrade organophosphates, the authors felt that an isolated enzyme would be less disruptive to the environment than would genetically altered microorganisms because the enzymes alone can not reproduce. Copella et al. (1990) found that the organism-free enzyme was able to hydrolyze the organophosphates. In another experiment, parathion hydolase was used in the cattle-dipping process. The process involves dipping the cattle in a bath containing the insecticide coumaphos to protect the cattle from the fever tick. One problem with this process is the formation of potassan (a compound toxic to cattle) from the breakdown of coumaphos. Parathion hydolase was found to selectively destroy potassan without degrading the parent compound of coumaphos and extending the time the dip could be used before it was no longer effective or became dangerous (Smith et al., 1992).

Use in Waste Treatment

Biocatalysts also have been used in conventional waste treatment processes. Enzymes have the potential applicability to be used in the treatment of cotton and bleach mills, as well as the destruction of particular compounds such as a variety of phenols and aromatic amines (Aitken, 1993). In one study (Kilbanov, 1980), the usefulness of the enzyme peroxidase extracted from horseradish, was evaluated for removal of phenols and anilines from wastewater. Although there are several chemical treatment methods available, such as extraction, absorption, chemical and biological oxidation, etc., there are problems with each including high cost, low efficiency, hazardous by-product formation, and incomplete purification. With the biocatalyst the phenols and anilines are transformed to a waterinsoluble polymer, which can be easily removed. Klibanov (1980) reported removal efficiencies as low as 63.3% for o-aminophenol, and as high as 99.8% for o-chlorophenol. Studies such as these help narrow the field of where certain enzymes are beneficial and when they are ineffective. Lagerkvist and Chen (1993) found that the addition of an enzyme product, known as Econase, resulted in enhanced degradation of municipal solid waste under both acidogenic and methanogenic conditions.

Enzyme inactivation will play an important role in determining the feasibility of enzymes in waste treatment applications, so it is important to study the mechanisms by which inactivation can occur. The first, thermal denaturation, is denaturation of proteins in an aqueous solution. This mechanism can be easily controlled by increasing the concentration of enzymes or by the addition of high molecular weight species to the mixture. Losing a prosthetic group that is non-covalently bonded can also inactivate enzymes. This phenomenon occurs when the pH is significantly out of range for the given enzyme. The problem can be alleviated with proper pH control. A third, less understood mechanism for inactivation, is phase transfer. In some experiments, the enzyme absorbed a polymeric oxidation product, making the substrate less viable to the solidified enzymes. The reaction products of the enzymes may need to be checked in any reactions where precipitate a is formed. The last inactivation pathway is mechanism-based inactivation. This occurs when a product formed from a reaction with an enzyme reacts with the enzyme in such a way as to inactivate it (Aitken, 1993). MATERIAL AND METHODS Experimental Design Anaerobic digestion is a process used in treatment of both high and low strength wastes. During anaerobic digestion, the microbial consortium breaks down the waste into methane, carbon dioxide, and water. Experiments were performed in completely sealed 4-liter glass digesters with a waste input stream, an output stream, and a gas collection system. The performance of the anaerobic digestion process was studied to determine its effectiveness in degrading primary municipal sludge with a 3% volatile solids content. Parameters studied were total solids, total volatile solids, chemical oxygen demand, pH, temperature, and two methods of enumerating bacteria levels within the reactors. One method was a standard anaerobic plate count of samples taken from the reactors. The other method used was an indirect method of sampling facultative and aerobic organisms within the reactors using a device known as an HMB-IVT~ from Biotech International.

 In the experiment twelve 4-liter reactors were maintained with an operating volume of 3-liters of municipal sludge from the Lubbock municipal wastewater treatment plant. Each reactor (Figure 1) was operated 9 months to achieve equilibrium before the commercial products were added. Once equilibrium had been attained, each of the five commercial products were fed into paired reactors for duplication of the results; two of the reactors were controls where no additive product was included (Table 1).

Table 1. Experimental Design of the Tests of Commercial Products Added to Anaerobic Digesters.

Reactor	Commercial Additives	Suppliers-location
R-1 & R-2	Bicope™	by Bicope Inc. Canyon, TX
R-3 & R-4	Medina depart	by Medina Agricultural Products Inc. Hondo, TX
R-5 & R-6	MPC	by Chemtecof Iowa.Des Moines, IA
R-7 & R-8	Alken Clear Flo 4100	by Alken Muray Corporation   New York, NY
R-9 & R-10	Biosep	by Sherman Enterprises   Lubbock, TX
R-11 & R-12	Control

effectiveness in degrading primary municipal sludge with a 3% volatile solids content. Parameters studied were total solids, total volatile solids, chemical oxygen demand, pH, temperature, and two methods of enumerating bacteria levels within the reactors. One method was a standard anaerobic plate count of samples taken f rom the reactors. The other method used was an indirect method of sampling facultative and aerobic organisms within the reactors using a device known as an HMB-IVm from Biotech International.

Figure 1. Schematic diagram of reactors used in these experiments.

The manufacturer's recommended dose determined the volume of product used in each reactor. All twelve reactors were placed on an oscillating shaker table that mixed all the reactors at a consistent rate. All of the reactors were operated at room temperature with no external heating and maintained at a temperature of approximately 23 °C + 2.5 °C. Each reactor consisted of a 4-liter glass aspirator type of bottle with an effluent port at the bottom for discharge and a rubber stopper sealed with silicon to prevent air from leaking into the system at the top (Figure 1). Each stopper contained two tubes. The first was a glass tube that extended well into the liquid layer of the reactor to keep gas from escaping. This tube had a funnel attached on the exposed end used for loading the reactors. The second tube extended only into the headspace of the reactors and was used for gas collection and analysis. The gas tube was attached to a collection bag that stored the gas until the volume was measured. Each line also had a Tjoint with a rubber septum attached in order to draw samples of gas to be analyzed in a gas chromatograph. Loading was performed on the reactors on a daily basis. The desired sludge retention time was 20 days, based on the optimum retention time found by Fedler (1985). For a retention time of 20 days, and a working volume of 3000 ml, the required flowrate was calculated to be 150 ml per day. Each day 150 ml of effluent were extracted for sample analysis and 150 ml of influent waste added to operate the reactors essentially as a continues stirred tank reactor (CSTR). Commercial Products Each of the five products was added daily with the influent waste. The products used in this study were BioCope~, Alken Clear-Flo09 4100, MPC, Medina d-part, and Biosep. Biocope™ is a liquid "multi-enzyme acting product" that will produce a floccueant mass of microorganisms that breaks down the waste into substrate for the organisms and oxygen to bring about an aerobic environment (Biocope, Inc., 1993). Although designed for aerobic treatment systems, BiocopeT~ was found effective for degrading cattle waste in an anaerobic system (Krishnamurthy, 1995).

Alken Clear Flow 4100 is designed to metabolize and control odor in agricultural waste pits and lagoons. It is a mixture of enzymes, emulsifiers, and specially adapted strains of bacteria and fungi (Alken Murray Corp., 1994). MPC is not an enzyme, but a mixture of a chemical emulsifier and O-dichlorobenzene that liquefies organic matter and controls odors (Chemtec, 1995). Medina d-part is a mixture of bacteria derived from a fermentation process. Medina's product is designed to degrade the waste in an aerobic environment while reducing grease, fats, and oils, reducing BOD and TSS levels and reducing odors (Medina). The manufacturers of Biosep refer to it as a "Hog Waste Manager," used for the treatment of nurseries and pit and lagoon treatments (Sherman, n.d.). It should be noted that none of these products have been manufactured specifically for use in anaerobic digesters, only for enhanced waste treatment in general.

Sample Collection and Measurement

Primary sludge was collected from Lubbock Municipal Waste Treatment Plant. Fifteen gallons of sludge was collected at one time to allow for a uniform mixture. The sludge was stored in a freezer until it was needed for loading into the digester. Volatile solids (VS) tests were run on the waste prior to use to determine the dilution needed to achieve a 3% volatile solids content in the influent. All tests performed on the reactor's effluent were on a weekly basis, except for pH and temperature, which were on a daily basis. The two tests for measuring the change in the microbial populations used were an anaerobic plate count and a test of aerobic organisms using the HMB-IV ™ developed by Biotech International.

For the anaerobic plate counts, the waste was uniformly distributed on Remel anaerobic blood agar plates using a Witley Automated SpiroplaterŪ (WASP) developed by Don Whitley Scientific Limited. The spiroplating method for enumeration of microorganisms was first introduced by Dr. J. E. Campbell in 1973, (Don Whitley Inc., 1995). This method has been documented as a viable alternative to conventional plate count techniques. It takes far less time to prepare plates, and reproduces results correlatable to standard techniques (Gilchrist et al, 1973). The waste was diluted to 1:10,000 to obtain countable colonies on the blood agar plates. The plates were then placed in sealed anaerobic bags from Becton Dickinson, which created an anaerobic environment. The bags were incubated in a 30°C incubator for 48 hours. The plates were then read using a Protos Colony Counter produced by Synoptic Ltd. This machine is equipped with a camera that takes images of the plate and transfers the image to a video screen. By setting the aperture and the shading of the colonies on the plate, the device calculates the colony forming units per milliliter of waste using a standard spiral plate counting method (Synoptic, Ltd., 1992).

The HMB-IV by Biotech International tests for aerobic and facultative biological activity within a liquid sample. The machine is based on the principle that most of these organisms produce catalase while they are active Aerobic organisms produce catalase to bind up free radicals of oxygen that can be destructive to microorganisms. Hydrogen peroxide is the oxidizing agent added to a sample. By adding the HMB-IV oxidizing agent to the waste in a sealed test tube, oxygen will be produced from the chemical reaction between the catalase and the hydrogen peroxide. Hydrogen peroxide will also bind with free iron. A chelating agent is added to bind with the free iron so that it does not interfere with the test (Biotech International). The machine measures the amount of oxygen that is produced, by the change in the pressure of the headspace of the test tube over a period of 15 minutes (U.S. Patent 4281536). Waste was diluted 1/100 for sampling purposes on the HMB-IV unit. Care must be taken in this experiment to follow the same procedure each time, because the amount and force of mixing can change the results.

Other tests that were run to determine effectiveness of the reactors included pH, temperature, chemical oxygen demand (COD), and total and   volatile solids. COD was analyzed with a calorimetric method using a HACH Spectrometer and parameters from Standard Methods (APHA, 1992). Allied studies not part of this project included the determination of gas composition and volume, fatty acid contents, and nitrogen levels within the reactor.

RESULTS AND DISCUSSION

System Start-up

Startup began by slowly feeding the reactors over several weeks until the volatile solids content of the influent was three percent. This was done to allow the microbial populations in the digesters to acclimate. Initially, two reactors were treated with the Biocope ™ additive to better approximate steady state conditions in reactors with an additive compared to those without additives. The remaining products were added to the reactors within the next two months.

Conventional Data Analysis

The most important test in determining the effectiveness of anaerobic digestion is the breakdown of total volatile solids (TVS) and total solids (TS). Overall only reactors receiving the Biocopew additive were significantly different from the control reactors, in terms of both TVS and TS concentration in the effluent (Figures 2 and 3). TVS and TS levels in these reactors were 16.5%, and 15% less than the control, respectively. Overall there was a 57.6% reduction of TVS and a 49.4% reduction of TS in reactors receiving the Biocope™ additive. Reactors receiving the MPC additive were the only ones that exhibited a significant (P<0.05) decrease in pH (Figure 4) or the biomass readout of the HMB-IV. There was no significant (P<0.05) difference in the COD values or the anaerobic plate count values of the effluents from any of the paired reactors.

Statistical analysis over the last two and one-half months of operation exhibited some results that were slightly different from the results of the entire operation of the experiment. The reactors receiving the Biocope additive were still the only ones with a significant increase in TS reduction, which averaged 10.2%, lower than the control. The reactors receiving the Biocope and MPC additives were the only reactors where there was a significant (P<0.05) difference in TVS degradation from the control. There was 11.3% greater breakdown of TVS than the control for a total breakdown efficiency of 54.0% in the reactors receiving the Biocope additive. The TVS breakdown of the reactors receiving the MPC additive was 12.5% less than the control for an overall TVS breakdown of 41.6%. This decrease in TVS degradation is probably a result of the subsequent drop in pH of the reactors receiving the MPC additive. The pH over this range was significantly different than the control reactors for reactors receiving MPC, Alken Clear Flo 4100@, and Biosep additives. The average pH of the effluent from the reactors receiving the MPC additive was 6.2, the only reactor pair that fell below the optimum pH range for anaerobic digestion. The reactors receiving the MPC additive were also the only reactor pair that was significantly (P<0.05) different than the control for the HMB-IV biomass readout. The reactors receiving the MPC additive had a biomass readout that was 58.0% lower than the control. The reactors receiving the MPC additive were also the only reactors where the reduction in COD were significantly (P<0.05) different from the control reactors. The average COD value in the effluent of the reactors receiving the MPC additive were 29.6% greater than the control.

The anaerobic plate count results of the reactors receiving MPC were the only tests that were significantly (P<0.05) different from the control. The average anaerobic population for the reactors receiving the MPC additive were 63.5% less than the control. It is unsure what caused the instability of the reactors that received the MPC additive. When the pH originally began to fall in the reactors receiving MPC, there was also a slight decrease in the pH of all the reactors including the control. This drop in pH was possibly due to an inhibitory product in the waste influent. The pH values of the reactors without MPC eventually stabilized or returned to their normal values, but the pH values of the reactors receiving MPC continued to decline. It is possible that the MPC additions caused a shift in the production of the natural buffering agents such as ammonium or bicarbonate. When a shock from an inhibitory product hit the reactors, the reactors receiving the MPC additive were not able to stabilize like the other reactors and the pH continued to drop.

Biological Evaluations

Experiments with the HMB-IV machine have shown it may be of some importance in maintaining anaerobic digesters in the proper pH range. It was observed that before the pH began to significantly drop in a reactor, the biomass readout would significantly drop. The drop in biomass readout would occur approximately 1 to 2 weeks before the subsequent drop in pH (Figure 5). An HMB-IV value of approximately 150 or below for these experiments correlated well with the drop of pH below 6.5. This trend needs to be analyzed further in future experiments to determine how effective a decrease of the aerobic microorganism activity can predict a decrease in the pH. This correlation between the HMB-IV biomass readout and pH could help operators stabilize the pH in reactors about to fail with small additions of bicarbonate before the pH ever begins to fall below the critical limit.

There was a difference between the MPC reactors and the control for the anaerobic plate count results. These findings support the literature's claim of the minimum ptI tolerance of approximately 6.5 (Miholits, 1965) for anaerobic microorganisms. The anaerobic plate count results also gave an indication of the total number of anaerobic organism within the digesters under normal conditions; approximately 1-2*107 anaerobic microorganisms per ml. The results show that other than MPC, the biocatalyst did not have a significant (P<0.05) effect on the anaerobic population within the digesters. Future experiments should be setup to examine the populations of organisms of each of the three different stages of anaerobic digestion, to see if the numbers are shifted towards the acetogenic or the methanogenic organisms by the introduction of the biological catalysts. Volatile fatty acid analysis and analysis of the gas produced might also help in identifying shifts within the reactors when compared to microorganism counting techniques.

A preliminary cost analysis was also performed to determine the possible savings when using the additives in a municipal wastewater treatment plant. Since Biocope™ was the only additive that showed a significant difference from the control reactors, it was used in this first level cost analysis. Since volatile solids destruction was 8.2% more than the control for the reactors receiving Biocopew, the cost analysis was performed on a typical 1 MOD over a 20-year lifetime wastewater treatment plant versus a treatment plant 8.2% smaller but receiving Biocope™ added per the manufacturer's recommendations. The cost analysis revealed a 1.90 benefit to cost ratio, with an overall savings of $193,257 over the 20-year lifetime of the plant. This was just a preliminary, Level 1 study, and more detailed analyses should be run to determine the actual benefit the additive would have on each piece of equipment in the treatment stream. This cost analysis did not consider other possible economic variables, such as, equipment and labor needed to adequately implement the additive to the treatment stream.

Figure3. Average totals solids values for all paired reactors(R1-R12) and the control. Additive in reactor indicated on legend.

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