Enzymatic monitoring and control of a two-phase batch digester leaching system with integrated anaerobic filter
© Gasch et al.; licensee Springer. 2013
Received: 6 May 2013
Accepted: 6 May 2013
Published: 29 May 2013
Biogas is one of the most promising regenerative energies. The simple fundamental principle underlying behind biomethanation is the conversion of biomass into biogas by microorganisms in four steps, whereby proper process regulation is crucial. Even today, the control of biogas plants is a challenging process which is based merely on physical and chemical parameters. A lack of meaningful microbiological control variables that can be quickly and easily determined locally makes it difficult for operators to react immediately if necessary.
In this study, different chemical parameters and several enzyme activities as well as their response to process disturbances were analyzed using two-phase anaerobic digestion systems with maize silage as substrate. The pH value, soluble chemical oxygen demand (COD), volatile fatty acids and total inorganic carbon (VFA/TIC) ratio, and biogas composition were examined using standardized cuvette or titration tests and online monitoring equipment. Hydrolase activities of esterase, amylase, protease, alanine-aminopeptidase, carboxymethyl cellulase, and xylanase were determined photometrically.
Hydrolases (esterase, amylase, protease, alanine-aminopeptidase, carboxymethyl cellulase, and xylanase) represent key enzymes in the first stage of biogas generation. In normal operation, the optimum values of the specific hydrolase activities varied over retention time. This allows for a visualization of the digestion progress. Furthermore, sudden strong activity attenuation has always indicated a disruption, even before any alterations in chemical parameters were affected. Beyond that, a direct correlation between esterase activity and COD discharge could also be identified. Moreover, in terms of the utilized substrate, specific microbial enzyme activities could be discovered as quality indicators. Thus, correlations to both the age of the silage and the effective cumulative COD yield could be determined.
Based on our analyses, the significance of various microbial enzyme activities and their association with the biogas process was demonstrated. The photometric determination of these enzyme activities allowed a sophisticated control of biomethanation to be carried out, which requires very little effort and equipment.
KeywordsAnaerobic digestion Two-stage Biogas Enzyme activity Hydrolases Monitoring Correlation
Currently, the state-of-the-art energy supply is primarily carried out with fossil fuels such as oil, coal, and natural gas. In the light of limited fossil energy reserves, it is necessary to develop and improve new sustainable and future-oriented concepts of energy supply. Therefore, the proportion of renewable energies such as biogas has to increase continuously . Biogas production is based on the technical usage of microbial activities, with the aim to convert organic substrates into methane as efficiently as possible. The anaerobic digestion is divided into four sub-processes - hydrolysis, acidogenesis, acetogenesis/dehydrogenation, and methanogenesis - which are characterized by different groups of microorganisms. By converting the products of the respective previous sub-process, they link the whole process . During the first step, complex organic molecules (polysaccharides, proteins, and lipids) are broken down into simpler organic molecules (sugars, amino acids, and long chain fatty acids). These reactions are catalyzed by hydrolases, which are secreted by hydrolytic bacteria into the environment. Acidogenic microorganisms transform these molecules into various intermediary products (low molecular organic acids, acetate, H2, and CO2). The processes of hydrolysis and acidogenesis cannot be clearly delineated as they typically proceed together in metabolism . Acetogenic bacteria convert the higher volatile fatty acids into acetate and hydrogen. Since the acetogenesis is only possible at a very low partial pressure of hydrogen, this partial step is coupled narrowly with methanogenesis. In this last step, two groups of methanogenic archaea produce methane from acetate or hydrogen and carbon dioxide. The first and second groups of microbes, as well as the third and fourth groups are linked closely with each other and have different requirements on the environment . In the two-stage biogas plants, the biogas process is divided into hydrolysis and methanation stages by spatial separation so that these different compulsory conditions can be better adjusted to shorten the treatment time. Therefore, two-stage anaerobic plants belong to the more effective systems for the conversion of solid substrates into biogas [4, 5]. However, a disadvantage is the complicated operation and control of the process . Monitoring of such biogas plants only by chemical and physical variables is apparently not sufficient. Thus, the efficiency of numerous plants is not satisfactory or even indeterminable . Therefore and because biogas production is a biological process, the analysis of microbiological parameters is very important. When the substrate to be fermented consists primarily of heavily degradable herbal components, the hydrolysis represents the rate-limiting step . Hence, control of this process stage is expedient. In several recent studies, the separation of the anaerobic digestion process into hydrolysis and methanogenic phases has been investigated with respect to hydrolases acting on solid biomass during anaerobic digestion [8–10]. Hydrolases are enzymes that catalyze the hydrolytic cleavage of a compound with the consumption of water. They are more or less strongly substrate specific. Therefore, the hydrolases that hydrolyze the abundant and poorly degradable substrate components are of special interest. In the case of herbal substrates, primarily, the activities of pectinase, amylase, cellulase (filter paper cellulase, β-glucosidase or carboxymethyl cellulase), xylanase, and protease were examined which catalyze the cleavage of pectin, starch, cellulose, xylan, and protein. In the analysis of hydrolase activity, Zhang as well as Parawira et al. [8, 10] identified the activity of free enzymes to be higher than the activity of cell-bound enzymes. This can be explained with the degradation behavior of particulate material. Compared to cell-bound enzymes, excreted enzymes have higher degradation efficiency  and seem to be significantly involved in the first step of the biogas process. Thus, they are responsible for the entire process speed and success. Further studies have even shown that the decomposition of structural cell components can be improved by the addition of hydrolases to the fermenter, reducing the digestion period possibly by about 30%;. Furthermore, the biogas yield was determined to increase up to 20%; [12–14]. For an analysis of the general heterotrophic degradation activity in biological systems, the determination of the esterase activity has been proven in practice. This holds true particularly for wastewater and soil analytics [15, 16]. For monitoring biogas plants, this analytical method has not been applied until now, although several relationships between the esterase activity and other process parameters could be identified in other bioengineered plants [17, 18]. This makes the esterase activity also interesting for the analysis and monitoring of biogas plants.
Here the microbiological analysis of two-phase anaerobic digestion systems by different enzyme activities of hydrolases (esterase, amylase, protease, alanine-aminopeptidase, carboxymethyl cellulase, and xylanase) is reported using simple photometrical assays as well as the correlation between selected chemical parameters and the entire biogas process.
Design of the study
Samples from percolators (solid samples), their direct effluent and samples from basins 1 and 2 were analyzed to detect the enzyme activities during the study period. Several chemical parameters including pH value, soluble chemical oxygen demand (COD), and volatile fatty acids and total inorganic carbon (VFA/TIC) ratio (content of volatile fatty acids to total inorganic carbon) were also measured in the samples. The method employed for COD measurements was analogous to EPA 410.4, US Standard Methods 5220 D and ISO 15705. VFA and TIC were measured using a two-step titration method . The methane content of the methane reactors was determined using both a gas flow meter (fluidistor-gas flow meter GD 100, Esters Elektronik, Rodgau, Germany) to detect the volume and a multichannel analyzer (multichannel analyzer biogas 905, ADOS, Aachen, Germany) to examine the components of the biogas.
For analysis of the biofilm of solid samples, the biofilm had to be separated from the substrate. For this, 20 g of solid sample was mixed with 200 mL of 0.9%; (w/v) NaCl solution and shaken vigorously for 1 min by hand or incubated for 2 h on a horizontal shaker (150 rpm, Ströhlein Instruments HS 500, Kaarst, Germany) or sonicated for 3 min in an ultrasonic bath with ultrasound frequency of 35 kHz (Sonorex Super RK 103H, Bandelin Electronic, Berlin, Germany). After allowing the solid particles to settle for some seconds, the supernatant was used for further analysis.
For the investigation of the removed biofilm from the substrate with regard to the efficiency of the methods, the total cell count of the supernatant was determined. The supernatants were fixed with a final concentration of 10%; (w/v) sodium azide (VWR, Prolabo, Fontenay sous Bois, France) for at least 2 h at 4°C, washed with phosphate-buffered saline (130 mM NaCl, 10 mM Na2HPO4/NaH2PO4, pH 7.4) at 10,000 × g for 10 min, resuspended in phosphate-buffered saline, and fixed by addition of ethanol (50%; v/v) for long-term fixation. The cells were stained using 4′,6-diamidino-2-phenylindole (Merck, Darmstadt, Germany) with a final concentration of 10 μg/mL for 15 min and homogenized by sonification (Sonopuls HD2070/SH70G with probe MS73, Bandelin Electronic, Berlin, Germany). After filtration of the samples (Isopore GTBP membranes, 0.2-μm pore size, Millipore, Eschborn, Germany), the filters were mounted in Citifluor (Citifluor Ltd., Canterbury, UK) and examined by using an epifluorescence microscope (Axioskop, Carl Zeiss, Jena, Germany) equipped with the filter combination: exciter filter 360/40, dichroic mirror 400 DC LP, and emission filter D 460/50 D (Carl Zeiss, Jena, Germany). For statistical analysis, at least ten randomly selected microscopic ocular grid fields were examined for each sample.
Enzyme activities were determined by centrifuging 10 mL of liquid sample at 13,000 × g for 10 min. The supernatant was collected and kept on ice until use for free enzyme activity assays. The determination of all enzyme activities was carried out in triplicate, and sample blanks were taken for each sample. Photometric measurements were performed on a Hitachi U-2000 spectrophotometer (Hitachi, Tokyo, Japan). The enzyme activity was expressed in international units (IU) - micromoles of substrate transformed per unit enzyme per minute and per milliliter sample or per gram fresh weight (FW) of substrate - unless otherwise stated.
The total activity of heterotrophic microorganisms was measured based on the method of Obst by the determination of the esterase activity . Supernatant with the amount of 0.5 mL was made up to a total volume of 9.9 mL with 60 mM sodium phosphate buffer (pH 7.6). After the addition of 100 μL substrate solution and 2 mg/mL fluorescein diacetate in acetone (Fluka, Buchs, Switzerland), the tubes were incubated on a shaker for 60 min at room temperature. After 10 min centrifugation at 4°C and 10,000 ×g, the absorbance of the supernatant was immediately measured photometrically against the sample blank at 490 nm. The esterase activity was calculated using a previously established standard curve with a fluorescein concentration range of 1 to 32 μmol/L (Fluka, Buchs, Switzerland).
Total protease activity was measured using the method of Mshandete et al. . Supernatant with the amount of 1 mL was added to 1 mL preheated 0.5%; (w/v) azocasein (Fluka, Buchs, Switzerland) in 200-mM Tris–HCl buffer (pH 7.4) and was mixed. After 1 h incubation at 50°C in a water bath, the reaction was stopped by adding 2 mL of 10%; (w/v) trichloroacetic acid. After 10 min of centrifugation at 4°C and 10,000 × g, the absorbance of the supernatant was measured photometrically against the sample blank at 380 nm. The protease activity was calculated using a previously created standard curve with the enzyme papain (Merck, Darmstadt, Germany) within a concentration range of 0.16 to 24 mg/L. Therefore, 1 mg/L papain corresponds to 30 USP units. Since in most cases the USP unit is equal to the international unit or IU, the term IU was also used.
l-Alanine-aminopeptidase activity was measured using the method of Remde and Tippmann . Supernatant with the amount of 2 mL was added to 2 mL of 0.1%; (w/v) l-alanine-4-nitroanilide hydrochloride (Merck, Darmstadt, Germany) in 0.9%; (w/v) NaCl and was mixed. After 1 h of incubation at 30°C in a water bath, the reaction was stopped by adding 2 mL of 10%; (w/v) trichloroacetic acid. Following a 10-min centrifugation at 10,000 × g, the absorbance of the supernatant was measured photometrically against the sample blank at 405 nm. The l-alanine-aminopeptidase activity was calculated using a previously prepared standard curve with a 4-nitroaniline concentration range of 0.16 to 24 mg/L (Merck, Darmstadt, Germany).
Hydrolase activities of amylase, carboxymethyl cellulase, and xylanase were examined by measuring the total reducing sugars produced using dinitrosalicylate reagent method based on Miller . Soluble starch of 1%; (w/v) (Merck, Darmstadt, Germany) was employed as a substrate for amylase, 1%; (w/v) carboxymethylcellulose (Fluka, Buchs, Switzerland) for carboxymethyl cellulase, and 0.5%; xylan from birch wood (Fluka, Buchs, Switzerland) for xylanase. All substrates were dissolved in 250 mM potassium phosphate buffer (pH 6.5). Supernatant with the amount of 150 μL was added to 350 μL of the pre-warmed substrate solution and mixed. After 1 h of incubation at 55°C in a water bath, the reaction was stopped by adding 750 μL dinitrosalicylic acid reagent (1%; (w/v) dinitrosalicylic acid, 0.2%; (v/v) phenol, 0.05%; (w/v) sodium sulfite, 20%; (w/v) potassium sodium tartrate, 1%; (w/v) NaOH). After 15 min incubation at 99°C in a thermomixer (Eppendorf, Hamburg, Germany), the tubes were set on ice for 15 min. The tubes were then centrifuged for 5 min at 10,000 × g, and the absorbance of the supernatant was measured photometrically against the sample blank at 575 nm. The enzyme activity was calculated using both a previously established standard curve with d-glucose (Carl Roth, Karlsruhe, Germany) for the determination of the amylase and cellulose activity and a previously established standard curve with d-xylose (Adlersdorf, Berlin, Germany) for the determination of the xylanase activity in a concentration range of 3 to 20 μmol/mL.
Results and discussion
Detachment of the biofilm from the substrate
To avoid interferences during the analysis of enzyme activities caused by the components of the substrate, a method had to be developed to separate the biofilm from the maize silage. The detachments obtained by vigorous shaking by hand, a two-hour shaking on a horizontal shaker, and a sonication method were tested. Since the detached biofilm would be used in this study to analyze the microbial activities, the total cell count was recorded to determine the efficiency of the methods. About 9 × 1010 cells/g fresh weight of substrate could be removed by manual shaking, 21%; more than by shaking horizontally, and 10%; more than by sonication. Therefore, this method was used for further analyses.
Analysis of the hydrolysis process
As previously mentioned, the enzyme activities of hydrolyzate do not correspond to those in the percolator. In the first 10 days, a lot of hydrolases were washed out, possibly without working in the percolator. Thus, for a detailed analysis of the degradation processes in the percolator, an investigation of the biofilm on the substrate is inevitable.
Visualization of disruptions in the process
Summary of significant correlations and corresponding statistical values
Sample size ( n)
Correlation coefficient ( r)
Correlation coefficient squared (r2)
Esterase activity (IU/mL)
Basin 1 + 2
Methane content (vol.%;)
2.44 × 10−4
Effluent of the percolator, basin 1 + 2
Effluent of the percolator, basin 1 + 2
1.64 × 10−50
Age of the silage (month)
6.28 × 10−4
Age of the silage (month)
4.35 × 10−2
Cumulative COD yield (mg/L)
Effluent of the percolator
5.76 × 10−3
Cumulative COD yield (mg/L)
Effluent of the percolator
2.43 × 10−2
The energy content of a substrate, and thus the expected theoretical methane yield, can be determined using the COD. Hence, it can be considered as an important process parameter . COD is the chemical oxygen demand required for the chemical conversion of the organic carbon compounds contained in the process water. These organic carbon compounds must be released first from the solid substrate by microorganisms. Therefore, the relationship between COD and the microbial activity is also interesting. In several experiments, we observed the anaerobic digestion systems I and II and analyzed the process water in terms of esterase activity and COD. As shown in Table 1, there exists a significant correlation between the esterase activity of the process water (direct effluent of the percolators, samples of basins 1 and 2) and the COD of the corresponding samples (r2 = 0.71; p value = 1.64 × 10−50; n = 184). The higher the esterase activity, the higher the COD. Moreover, it has been discovered that in comparison to the amount of esterase activity, a particularly high COD is discharged from the percolators within the first 3 days of fermentation. Therefore, within this period an esterase activity of 1 IU/mL corresponds to a COD discharge of approximately 25,000 mg/L. When all measured values are included, except the days 0 to 3 of the direct effluent of the percolators, an esterase activity of 1 IU/mL corresponds to a COD concentration of approximately 11,300 mg/L. An explanation for the high COD values in comparison to the esterase activity in the effluent of the percolators in the first days of fermentation could be an increased acid concentration. Thus, the average pH value in this phase of 5.6 was significantly lower than the average pH of the remaining process water with a value of 7.5. The VFA/TIC ratio also tended to be very high during the first days (Figure 2). This can be explained by the availability of easily degradable material in the fresh silage and a resulting strong production of organic acids like lactic acid, acetic acid, n-valeric acid, propionic acid, n-butyric acid, and other organic acids during the first few days [3, 31]. In addition, in the first days, the organic acids produced by ensiling (mainly lactic acid) are washed out. Since nonspecific esterases show a strongly reduced activity at pH values below 6 [32, 33] in this phase, the ratio of esterase activity and COD is shifted. However, in general a high esterase activity in the influent of the methane reactors is an indication of a high COD and a high expected theoretical methane yield. However, a high COD in the methane reactor effluent, as compared to the COD of the influent, suggests both that it could not be converted in the reactor to methane and that there are disturbances.
Since amongst others the quality of the input is crucial for the efficiency of the whole process, the fresh maize silage was also analyzed with regard to the possible indicative parameters. The maize silages analyzed here were derived from different charges of similar good quality. Interesting relationships could be detected. As presented in Table 1, there exists a negative correlation between the enzyme activities of esterase and aminopeptidase to the age of the maize silage (r2 = 0.79; p value = 6.28 × 10−4; n = 10 respectively r2 = 0.68; p value = 4.35 × 10−2; n = 6). The fresher the silage, the higher the enzyme activities. The principle of the ensiling is based on a fermentation process in which anaerobic microorganisms form acids, preferably lactic acid, and the pH value is lowered. As a result, cell respiration, butyric acid production, and protein breakdown are minimized. Thus, under these conditions, the metabolic process is greatly limited by self-inhibition of the microorganisms and the biomass is preserved for a longer time . Therefore, enzyme activity decreases depending on the storage time of the silage. If the silage is several months old and has a low enzyme activity, this points to a good quality of silage with a minimum energy loss by microbial degradation. However, when the storage conditions are poor, for example, the substrate comes in contact with oxygen which can cause a secondary aerobic decomposition , the microbial activity would be high.
The control of the biogas process begins with the selection of a suitable substrate. Maize silage is still a very common substrate exhibiting high biochemical methane potential. The energy content is highly dependent on the ensiling quality. In this study, it was demonstrated that the enzyme activities of esterase and aminopeptidase permit conclusions about the age of maize silage and consequently the success of ensiling. In addition, a correlation between the aminopeptidase or cellulase activity and the cumulative COD yield has been found, which allows us to assess the biogas production potential. Thus, enzymatic analysis of the substrate permits an assessment of its quality as a biogas substrate. In two-stage biogas plants, the hydrolysis of herbal material is often the rate-limiting step. Hence, the analysis of this process phase is a possible monitoring tool. Therefore, analysis of the microbial activity is of particular importance. By determining substrate-specific hydrolases during the process, the hydrolysis progress can be observed. High xylanase and cellulase activities, for example, point to a very advanced degradation in herbal substrates. If the general microbial activity is monitored continuously, process disturbances can be detected by a drop in the measured values. A high correlation of esterase activity with COD supports this observation. Furthermore, this demonstrates the significance of this microbiological parameter for the biogas process.
The levels of enzyme activity can be useful for monitoring parameters during anaerobic digestion, since enzyme activities are relatively easy to measure. Moreover, it is possible to develop a simple assay that can be locally used by the operator of the biogas plant rapidly in a cuvette test. This would be a quick and inexpensive way to analyze the substrate and monitor the biogas process online and possibly to respond directly to detected process disruptions. As a consequence, monitoring of enzymatic activities enables a straightforward analysis of the biogas process, avoiding a tedious analysis of numerous chemical parameters. This allows for a novel and an extensive control of the biogas process, thus capitalizing on its full benefits.
Chemical oxygen demand
- D1 and 2:
Depth 1 and 2
Total inorganic carbon
Volatile fatty acids
The authors would like to thank Dr. Axel Wobus for the constructive advice in the enzymatic studies. They are also grateful to Dr. Kerstin Röske for the constructive criticism when designing this article. The authors would also like to thank the Boden- und Grundwasserlabor GmbH and the Großmann Ingenieur Consult GmbH for their financial support of the project.
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