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Anaerobic degradation of amino acids generated from the hydrolysis of sewage sludge a
a
Junghoon Park , Seyong Park & Moonil Kim
a
a
Department of Civil & Environmental Engineering, Hanyang University, 55 Hanyangdaehakro, Ansan, Gyeonggido, Korea Published online: 10 Dec 2013.
Click for updates To cite this article: Junghoon Park, Seyong Park & Moonil Kim (2014) Anaerobic degradation of amino acids generated from the hydrolysis of sewage sludge, Environmental Technology, 35:9, 1133-1139, DOI: 10.1080/09593330.2013.863951 To link to this article: http://dx.doi.org/10.1080/09593330.2013.863951
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Environmental Technology, 2014 Vol. 35, No. 9, 1133–1139, http://dx.doi.org/10.1080/09593330.2013.863951
Anaerobic degradation of amino acids generated from the hydrolysis of sewage sludge Junghoon Park, Seyong Park, and Moonil Kim∗ Department of Civil & Environmental Engineering, Hanyang University, 55 Hanyangdaehak-ro, Ansan, Gyeonggido, Korea
Downloaded by [McMaster University] at 07:38 20 December 2014
(Received 23 April 2013; accepted 30 October 2013 ) The anaerobic degradation of each amino acid that could be generated through the hydrolysis of sewage sludge was evaluated. Stickland reaction as an intermediate reaction between two kinds of amino acids was restricted in order to evaluate each amino acid. Changes in the chemical oxygen demand (COD), T-N, NH+ 4 -N, biogas, and CH4 were analysed for the anaerobic digestion process. The initial nitrogen concentration of all amino acids is adjusted as 1000 mg/L. The degradation rate of the amino acids was determined based on the ammonia form of nitrogen, which is generated by the deamination of amino acids. Among all amino acids, such as α-alanine, β-alanine, lysine, arginine, glycine, histidine, cysteine, methionine, and leucine, deamination rates of cysteine, leucine, and methionine were just 61.55%, 54.59%, and 46.61%, respectively, and they had low removal rates of organic matter and showed very low methane production rates of 13.55, 71.04, and 80.77 mL CH4 /g CODin , respectively. Especially for cysteine, the methane content was maintained at approximately 7% during the experiment. If wastewater contains high levels of cysteine, leucine, and methionine and Stickland reaction is not prepared, these amino acids may reduce the efficiency of the anaerobic digestion. Keywords: anaerobic degradation; amino acids; stickland reaction; deamination; biogas
Introduction The protein amounts in the organic content of sewage sludge range from 30% to 70%.[1] If 50% of that organic content is composed of protein, then the amino acids created from hydrolysis can also be primary ingredients in the anaerobic digestion process of the sewage sludge. However, only a few studies have looked into the amino acids that resulted from proteolysis, and most studies only focused on how the ammonia that results from the protein inhibits the activity of acidogenic bacteria and methanogens.[2–4] The process by which amino acids are converted into methane in anaerobic conditions has been identified through previous studies. Amino acids are amphoteric substances that contain amino and carboxyl groups, and they are the result of a four-step process of hydrolysis, amino acid fermentation, acid production, and methanation of the anaerobic degradation process of proteins.[5] The degradation products of these amino acids are organic compounds (short-chain and branched-chain organic acids), ammonia, carbon dioxide, and small amounts of hydrogen and sulphur compounds.[5] Amino acids are decomposed in two ways.[6] The first is deamination through a Stickland reaction, when two types of amino acids are injected. One side of the amino acid (containing the majority of the carbon atoms) acts as an electron accepter, and the other side (containing one or only a few carbon atoms) acts as an electron donor. The reaction that takes place is the deamination by bacteria ∗ Corresponding
author: Email: [email protected]
© 2013 Taylor & Francis
within the clostridium species (obligatory anaerobe).[6] It is known that these clostridium bacteria are unable to deaminate single amino acids.[7] The second type of amino acid decomposition occurs through the general fermentation process of single amino acids. Among these two types of the degradation, the fermentation of amino acids by the Stickland reaction is known to be the dominant reaction.[6] Elbeshbishy and Nakhla determined that a ratio of 8:2 (protein:carbohydrate) enables the most efficient anaerobic digestion when co-treating protein and carbohydrates.[8] Also, the amounts of organic matter removal and methanation decreased with higher protein content. In this case, it was reported that anaerobic digestion based only on protein was the least efficient. Another study reported that when protein was removed through an isoelectric point and anaerobically digested, the anaerobic efficiency increased. Also, if protein content increased, then the efficiency level decreased.[9] This result is due to the low C/N ratio, propionic acid accumulation, and hindrance of the microorganism activity due to the increase in the concentration of the ammonia-nitrogen, which was formed from proteolysis. However, Wagner and researchers stated that the methane production rate has no correlation with the carbon content in the anaerobic digestion of different kinds of protein sources and amino acids, and that the C/N ratio also had no effect.[10] It is difficult to correctly identify the methane production amount per organic compound removed since
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the latter was not taken into account when the reaction subsided. However, it is apparent that the anaerobic degradation efficiency of each amino acid that forms protein differs from those of the others. Therefore, for this study, we chose various amino acids [11,12] that are known to be created from the hydrolysis of sewage sludge and sorted them as neutral non-polar, neutral polar, alkaline, and acidic. Then, the anaerobic degradation process was evaluated in each group individually. In order to identify the degradation efficiency and the time required for the individual amino acids existing as organic nitric compounds to break down into volatile acid and ammonia, the T-N concentration of each amino acid was set at a fixed value. Also, the deamination of amino acids during the experiment increased the ammonia-nitrogen concentration, which made it possible to identify the degradation rate of amino acids. As the reaction proceeded, each item’s anaerobic resolution was identified through the change in its concentration and the methane production amount. Materials and methods Test equipment The Biochemical Methane Potential test [13] was used in this study in order to identify the anaerobic degradation process of each amino acid. For the experiment, we injected 25 mL of synthetic wastewater with amino acid and the same amount of sludge into a 125 mL serum bottle that was then placed in a thermostatic chamber at a warm temperature of 35◦ C. Before sealing the bottle, any internal oxygen was removed and plenty of nitrogen gas was injected in order to ensure that the sample was anaerobic. Nine serum bottles of the same size were made for each synthetic wastewater sample containing amino acid. A total of 90 bottles were used in the experiment including the nine control serum bottles. In order to examine the changes in the amino acids, chemical oxygen demand (COD), T-N, NH+ 4 -N, and pH during the reaction, samples were taken from each serum bottle and measured accordingly. The experimental accuracy was judged by the biogas production amount measured until two of the nine original serum bottles were left for each item. Table 1.
The gas emission measurements were taken using a manometer to measure the difference between the inner pressure of the bottle and the atmosphere. During the early part of the reaction, measurements were taken every 6 h. As the reaction time increased, measurements were taken at two and four days. The experiment was concluded when no more biogas or organic compounds remained to remove. The amount of methane produced was measured as shown below [14] VCH4 = C1 (V1 + V0 ) − C0 V0 , where VCH4 is the volume of methane produced (mL), C1 the methane content (%) at sampling time, C0 the methane content (%) at the previous sampling time, V1 the biogas volume measured by syringe (mL), and V0 the gas phase volume of the reactor (mL). Synthetic wastewater and inoculum Synthetic wastewater Synthetic wastewater, which consists of individual amino acids, is composed as given in Table 1. In order to determine the efficiency rate and time needed for each amino acid that existed in the form of organic nitrogen to decompose into volatile organic acid and ammonia (inorganic nitrogen), the synthetic wastewater was made by maintaining the T-N concentration of each amino acid at 1000 mg/L. Also, the degradation efficiency was identified through the increase in the concentration of ammonia-nitrogen caused by the deamination of the amino acid. Microelements that are known to positively affect microorganism activity was injected.[15] Microorganism seeding The microorganism that was used to identify the anaerobic degradation types of the individual amino acids was inoculated by granular sludge collected from a 35◦ C anaerobic reactor in Cheongwon-gun, Korea. The mixed liquor suspended solids (MLSS) concentration and the mixed liquor volatile suspended solids (MLVSS) concentration of the
Characteristics of each amino acid.
Name α-Alanine β-Alanine Lysine Arginine Glycine Histidine Cysteine Methionine Leucine
Molecular weight
Molecular formula
COD (mg/L)
T-N (mg/L)
NH+ 4 -N (mg/L)
pH
89 89 146 174 75 155 121 149 131
CH3 CH(NH2 )COOH NH2 CH2 CH2 COOH H2 N(CH2 )4 CH(NH2)COOH H2 NC=(NH)NH(CH2 )3 CH(NH2 )COOH CH2 (NH2 )COOH 2-NH2 -3(CH)2 N(NH)C-C2 H3 COOH HS-CH2 CH(NH2 )COOH CH3 S(CH2 )2 CH(NH2 )COOH (CH3 )2 CHCH2 CH(NH2 )COOH
6857.15 6857.15 6857.14 4428.56 3428.58 3809.53 11428.57 18285.71 17142.85
1000 1000 1000 1000 1000 1000 1000 1000 1000
0 0 0 0 0 0 0 0 0
6.93 6.99 6.86 9.39 7.03 7.46 6.58 7.02 7.04
Environmental Technology Table 2.
Characteristics of granular sludge.
Items MLSS MLVSS Soluble COD Soluble T-N Soluble NH+ 4 -N pH
Table 3. Increased T-N concentrations due to granular sludge degradation.
Concentration (mg/L) 19,900 18,500 47 21 20.25 6.93
granular sludge was 19,900 and 18,500 mg/L, respectively. Its characteristics are given in Table 2.
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Analytical methods The COD, MLSS, MLVSS, and T-N were determined according to Standard Methods.[16] The NH+ 4 -N was measured using a spectrophotometer (DR/2500, Method 8038, Nessler Method, Hach Co., USA). Only matters in a dissolved state were measured through filtering (0.45 μm) for COD, T-N, and NH+ 4 -N. A manometer was used to measure the amount of biogas production, and the methane content was measured through gas chromatography (Varian Model STAR 3400CX, USA; carrier gas, N2; injector temp, 150◦ C; column temp., 29◦ C; detector temp., 200◦ C). The pH was measured using a pH meter (Cyber scan pH 20). Results Nitrogen production caused by the hydrolysis of granular sludge T-N concentration within the serum bottle was 1000 mg/L. When 100% of the amino acid is deaminated, the ammonianitrogen concentration increases from 0 to 1000 mg/L. However, as the extracellular polymers (ECPs) from the granular sludge decomposed, the nitrogen concentration increased to a T-N concentration of over 1000 mg/L (1050– 1560 mg/L) approximately 25 days after the experiment. ECPs promote cell to be survived by protecting and maintaining the ectoenzyme of the cell surface. ECPs also act as ion-exchange resins that manage the ionic migration from the aqueous solution to the cell. Generally, ECPs are known to be primarily consisting of protein (52%), humus (41%), and carbohydrates (7%).[1] It was determined that the ammonia-nitrogen concentration reached over 1000 mg/L during the reaction because of the protein degradation (the major component of ECPs). The T-N concentration of each amino acid throughout the experiment increased more in the types of matter with low concentrations of COD injected or in those with less COD substance removal. It was because of the increase in cell lysis rate with insufficient degradable organics. It was estimated that the nitrogen increase in cysteine was higher than that the control without organic injection because a lot of cell lysis occurred when the cysteine worked as a toxicant
Increased T-N Concentration α-Alanine β-Alanine (mg/L)
50
50
Glycine 110
Histidine Cysteine 110 560
Lysine 60
Arginine 120
Methionine Leucine 240 170
for microorganisms. The increases in the nitrogen concentration for each amino acid are shown in Table 3. For clearer identification of deamination followed by the increase in the ammonia-nitrogen for each amino acid, granular sludge was injected into the control sample instead of the amino acids. Then, the control sample T-N and ammonia-nitrogen concentrations were measured according to the process of time. The results are shown in Figure 1. The increases in the T-N and ammonia-nitrogen concentrations followed a linear equation. The increase in the T-N concentration in the serum bottle was assumed to be caused by an increase in the nitrogen concentration resulting from the degradation of the ECPs. This increase is shown in Table 3. Through the increase in the T-N concentration, it was possible to calculate the ammonia-nitrogen concentration produced by the granular sludge. Then, the calculated value was subtracted from the measured ammonia-nitrogen concentration. The generation rate of the ammonia-nitrogen is given in Table 4. Ammonia-nitrogen production and COD removal Figure 2 shows the anaerobic degradation of each amino acid and the control sample (granular sludge without amino acids). The control sample had the same synthetic wastewater without the injection of any amino acids, and the increase in the nitrogen concentration caused by degradation of the ECPs was measured when no substrates remained for the microorganisms to use. As shown in (a)-(i) of Figure 2, the ammonia-nitrogen concentrations of glycine, lysine, α-alanine, histidine, and arginine increased rapidly for three days after the reaction but then began to decline. In the case of α-alanine, the concentration steadily increased until day 3 and showed a rapid increase between days 3 and 5. On the other hand, the ammonia-nitrogen concentrations of cysteine, leucine, and methionine showed steady increases with no rapid changes. Alanine is classified into α- and β-alanine according to the position of the amino groups within the molecular structure. These different structures cause different microorganism decay rates. A research by Wagner and others indicated that the individual peptone extracted from different protein ingredients shows different conversion rates under anaerobic conditions.[10] Methionine and cysteine are amino acids that deaminate through Stickland and non-Stickland reactions. Leucine
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J. Park et al. 500
500 T-N NH4+-N T-N NH4+-N
300
400
200
200
y = 9.9323x + 26.1016
100
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300
y = 12.6825x + 25.7561
0
0
5
10
15
20
T-N, mg/L
NH4+-N, mg/L
400
100
25
30
0
Day
Figure 1. Table 4. acid.
Nitrogen generation by granular sludge degradation. Generation rate of ammonia-nitrogen for each amino
NH+ 4 -N Generation (%) Glycine 91.71
α-Alanine β-Alanine 92.99 Histidine 91.34
92.99
Lysine
Arginine
91.21
94.01
Cysteine Methionine Leucine 61.55 46.61 54.59
deaminates through only the Stickland reaction.[6] The ammonia-nitrogen concentration of cysteine is higher than that of methionine due to the fact that while only C. propionicum deaminates methionine, cysteine is degraded by more types of microorganisms, which results in a higher concentration of ammonia-nitrogen. However, even though the generation rates of ammonia-nitrogen that were generated by the deamination of cysteine and methionine were 61.55% and 46.61%, respectively, no organic matter was removed. Table 4 shows the generation rates of the ammonianitrogen concentration (excluding the ammonia-nitrogen concentration from granular sludge). Although leucine is an amino acid that is known to only deaminate through the Stickland reaction,[6] this study found a deamination of 54.59% despite the limiting Stickland reaction. This result is due to the fact that as the granular sludge steadily decomposes during the reaction, it creates Stickland reaction conditions in which leucine can be decomposed. As shown in graph (j) in Figure 2, when there are no more substrates for the microorganism to use, it can be inferred that the ammonia-nitrogen concentration increased from 0 to 255 mg/L. As for cysteine and methionine, when serine exists under anaerobic conditions, it decomposes
into propionic acid and acetic acid through the Stickland reaction.[10] The deamination progress rates of these acids were 61.55% and 46.64%, respectively. Although this was an individual condition without serine, deamination was possible because the granular sludge undergoes the reaction and steadily decomposes; therefore, it cannot completely limit the Stickland reaction. Nevertheless, it is clear that the deamination of cysteine and methionine relies on the Stickland reaction, unlike the general fermentation process of the anaerobes of amino acids. Also, amino acids usually convert into volatile acids because the amino group detaches through deamination.[5] However, it was revealed that there are cases in which the organic carbon sources are not decomposed anaerobically in the case of cysteine and methionine. There could be a possibility that these amino acids could have been converted into propionate which is known as one of relatively hardly degradable volatile fatty acids. More research to clarify the deamination of cysteine and methionine is necessary. Other amino acids excluding cysteine, methionine, and leucine were not numerically recorded but showed greater than 90% ammonia-nitrogen generation and COD removal rates. Methane production according to the injected amount of COD Since the nitrogen concentration of each amino acid was fixed at 1000 mg/L, the level of injected COD was different, which made it impossible to compare the biogas production amount for each item. This is why the methane production rates per (g) COD input are calculated in Table 5. As noted in Figure 2, the methane production rates at the time of measurement were the highest between days 3
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Environmental Technology
Figure 2.
Anaerobic degradation of each amino acid.
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J. Park et al. Table 5.
CH4 generation from each amino acid.
Initial COD (mg/L) CH4 generation (mL) mL CH4 /g CODin
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Initial COD (mg/L) CH4 generation (mL) mL CH4 /g CODin
α-Alanine
β-Alanine
Lysine
Arginine
6333 159.03 381.82 Glycine 3506 104.13 424.2
6333 153.81 396.09 Histidine 3767 82.69 396.36
6240 123.73 300.07 Cysteine 10,620 10.11 13.55
3041 86.27 444.7 Methionine 12,350 51.95 71.04
and 7. The α, β-alanine had a methane production as high as 68.8%. The reason that the methane production levels were maximal and then slowly declined is because that the experiment was not continuous, but a batch experiment. In the case of leucine, which has high COD input and a low COD removal rate, the methane production rate was maintained at its highest number. As with cysteine, the methane production rate appeared to be lower than that of the control bottle. Although neither methionine nor cysteine showed signs of organic matter removal from deamination half-way through the experiment, methionine showed similar levels of methane production to the control sample. This result supported that cysteine acted as a toxic substance against methanogens. Arginine, glycine, and histidine had methane production rates per COD input of 443.70, 424.20, and 396.36 mL CH4 /g CODin , respectively, and these results were higher than those for alanine and lysine. A theoretical methane production of anaerobic digestion is 395 mL CH4 /g COD.[1] The reason why the methane production rate per COD input concentration of arginine, glycine, and histidine was higher than the theoretical amount is because biogas was produced along with an increase in the nitrogen concentration when the ECPs decomposed. As mentioned earlier, the nitrogen concentration increased significantly in matter without proper COD removal or at a low COD input concentration. Likewise, the T-N concentration of lysine and alanine increased 60 and 50 mg/L, respectively, when the reaction ended. On the other hand, arginine, glycine, and histidine showed increases in 120, 110, and 110 mg/L, respectively. Therefore, it can be inferred that as high as was the increase in nitrogen concentration from the granular sludge, the related biogas production rates would have been even higher. Cysteine, methionine, and leucine had low removal rates of organic matter and showed very low methane production rates of 13.55, 71.04, and 80.77 mL CH4 /g CODin , respectively. Especially for cysteine, the methane content was maintained at approximately 7% during the experiment, which was a lower value than in the control sample. Cysteine is known to decompose into acetic acid, ammonia, carbon dioxide, hydrogen sulphide, and hydrogen through the Stickland reaction.[6] In this study, cysteine did not decompose under anaerobic conditions and was found to limit methanation activation. Therefore, whether or not
Leucine 15,833 37.41 80.77
cysteine can be decomposed under anaerobic condition needs to be assessed under mixed conditions that involve the Stickland reaction. Conclusions The results of the anaerobic degradation estimation for individual amino acids in order to estimate the anaerobic digestion of the amino acids that are formed by the hydrolysis of sewage sludge are as follows: 1. More than 90% of α-alanine, β-alanine, lysine, arginine, glycine, and histidine was converted into ammonia-nitrogen due to the deamination of amino acids, but only 61.55%, 54.59%, and 46.61% of cysteine, leucine, and methionine, respectively, were converted into ammonia-nitrogen. 2. It has been found that cysteine, leucine, and methionine, which showed low production rates of ammonianitrogen, require the Stickland reaction as a necessary condition for complete degradation. If that condition is met, more research is required as to whether the degradation product, which is produced along with the ammonia-nitrogen, can be decomposed under anaerobic conditions. 3. Although the methane production rates according to COD input of arginine, glycine, and histidine were 444.70, 424.20, and 396.36 mL CH4 /g CODin , respectively, these values are higher than the theoretically estimated value. This result is due to the fact that the degradation of the granular sludge ECPs caused gas production. In turn, cysteine, leucine, and methionine, which had low levels of ammonianitrogen production, had very little organic matter removal. This also resulted in methane production rates per COD input of 13.55, 80.77, and 71.04 mL CH4 /g CODin , respectively, which is much lower than the rates for other amino acids. This study assessed the anaerobic digestion of individual amino acids. Although not all of the existing amino acids were assessed for anaerobic digestion, this study found that amino acids other than cysteine, leucine, and methionine were easily decomposed anaerobically. As for cysteine, leucine, and methionine, an assessment is necessary under
Environmental Technology mixed conditions and involving the Stickland reaction because they are not decomposed well under individual conditions. If these three amino acids are present in high concentrations in wastewater and do not satisfy the Stickland reaction, they will cause a less efficient anaerobic digestion process. Funding This research was supported by the Energy Non-CO2 Greenhouse Gas Group. We deeply appreciate their support [Project Number: 201200000001521].
[8] [9] [10]
[11]
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References [1] Speece RE. Anaerobic biotechnology and odor/corrosion control. Nashville, TN: Archae Press; 2008. [2] Sung S, Liu T. Ammonia inhibition on thermophilic anaerobic digestion. Chemosphere. 2003;53:43–52. [3] Zeshan OP, Karthikeyan CV. Effect of C/N ratio and ammonia-N accumulation in a pilot-scale thermophilic dry anaerobic digester. Bioresour Technol. 2012;113:294–302. [4] Gallert C, Winter J. Mesophilic and thermophilic anaerobic digestion of source-sorted organic wastes: effect of ammonia on glucose degradation and methane production. Appl Microbiol Biotechnol. 1997;48:405–410. [5] Barker HA. Amino acid degradation by anaerobic bacteria. Annu Rev Biochem. 1981;50:23–40. [6] Ramsay IR, Pullammanappallil PC. Protein degradation during anaerobic wastewater treatment: derivation of stoichiometry. Biodegradation. 2001;12:247–257. [7] Fonknechten N, Chaussonnerie S, Tricot S, Lajus A, Andreesen JR, Perchat N, Pelletier E, Gouyvenoux M, Barbe V, Salanoubat M, Le Paslier D, Weissenbach J, Cohen GN, Kreimeyer A. Clostridium sticklandii, a specialist in
[12]
[13] [14]
[15] [16]
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amino acid degradation: revisiting its metabolism through its genome sequence. BMC Genomics. 2010;11:5551–12. Elbeshbishy E, Nakhla G. Batch anaerobic co-digestion of proteins and carbohydrates. Bioresour Technol. 2012;116: 170–178. Chen Ye, Cheng Jay J., Kurt S. Creamer, inhibition of anaerobic digestion process: a review. Bioresour Technol. 2008;99:4044–4064. Wagner AO, Hohlbrugger P, Lins P, Illmer P. Effects of different nitrogen sources on the biogas production – a lab-scale investigation. Microbiol Res. 2011;167: 630–636. Orlygsson J, Houwen FP, Svensson BH. Influence of hydrogenothrophic methane formation on the thermophilic anaerobic degradation of protein and amino acids. Microbiol Ecol. 1994;13:327–331. Baena S, Fardeau ML, Labat M, Ollivier B, Garcia JL, Patel BK. Desulfovibrioaminophilus sp. nov., a novel amino acid degrading and sulfate reducing bacterium from an anaerobic dairy wastewater lagoon. Syst Appl Microbiol. 1998;21:498–504. Owen WF, Struckey DC, Healy JB, Young LY, McCarty PL. Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res. 1979;13:485–492. Nasr N, Elbeshbishy E, Hafes H, Nakhla G, Naggar MHE. Comparative assessment of single-stage and twostage anaerobic digestion for the treatment of thin stillage. Bioresour Technol. 2012;111:122–126. Kim M, Ahn Y, Speece RE. Comparative process stability and efficiency of anaerobic digestion; mesophilic vs. thermophilic. Water Res. 2002;36:4369–4385. American Public Health Association (APHA). The American Water Works Association (AWWA), and the Water Environment Federation (WEF), Solids, Standard Methods for the Examination of Water and Wastewater. 21st ed. Washington, DC: APHA; 2005, 2-55–2-60.
Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Anaerobic degradation of amino acids generated from the hydrolysis of sewage sludge a
a
Junghoon Park , Seyong Park & Moonil Kim
a
a
Department of Civil & Environmental Engineering, Hanyang University, 55 Hanyangdaehakro, Ansan, Gyeonggido, Korea Published online: 10 Dec 2013.
Click for updates To cite this article: Junghoon Park, Seyong Park & Moonil Kim (2014) Anaerobic degradation of amino acids generated from the hydrolysis of sewage sludge, Environmental Technology, 35:9, 1133-1139, DOI: 10.1080/09593330.2013.863951 To link to this article: http://dx.doi.org/10.1080/09593330.2013.863951
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Environmental Technology, 2014 Vol. 35, No. 9, 1133–1139, http://dx.doi.org/10.1080/09593330.2013.863951
Anaerobic degradation of amino acids generated from the hydrolysis of sewage sludge Junghoon Park, Seyong Park, and Moonil Kim∗ Department of Civil & Environmental Engineering, Hanyang University, 55 Hanyangdaehak-ro, Ansan, Gyeonggido, Korea
Downloaded by [McMaster University] at 07:38 20 December 2014
(Received 23 April 2013; accepted 30 October 2013 ) The anaerobic degradation of each amino acid that could be generated through the hydrolysis of sewage sludge was evaluated. Stickland reaction as an intermediate reaction between two kinds of amino acids was restricted in order to evaluate each amino acid. Changes in the chemical oxygen demand (COD), T-N, NH+ 4 -N, biogas, and CH4 were analysed for the anaerobic digestion process. The initial nitrogen concentration of all amino acids is adjusted as 1000 mg/L. The degradation rate of the amino acids was determined based on the ammonia form of nitrogen, which is generated by the deamination of amino acids. Among all amino acids, such as α-alanine, β-alanine, lysine, arginine, glycine, histidine, cysteine, methionine, and leucine, deamination rates of cysteine, leucine, and methionine were just 61.55%, 54.59%, and 46.61%, respectively, and they had low removal rates of organic matter and showed very low methane production rates of 13.55, 71.04, and 80.77 mL CH4 /g CODin , respectively. Especially for cysteine, the methane content was maintained at approximately 7% during the experiment. If wastewater contains high levels of cysteine, leucine, and methionine and Stickland reaction is not prepared, these amino acids may reduce the efficiency of the anaerobic digestion. Keywords: anaerobic degradation; amino acids; stickland reaction; deamination; biogas
Introduction The protein amounts in the organic content of sewage sludge range from 30% to 70%.[1] If 50% of that organic content is composed of protein, then the amino acids created from hydrolysis can also be primary ingredients in the anaerobic digestion process of the sewage sludge. However, only a few studies have looked into the amino acids that resulted from proteolysis, and most studies only focused on how the ammonia that results from the protein inhibits the activity of acidogenic bacteria and methanogens.[2–4] The process by which amino acids are converted into methane in anaerobic conditions has been identified through previous studies. Amino acids are amphoteric substances that contain amino and carboxyl groups, and they are the result of a four-step process of hydrolysis, amino acid fermentation, acid production, and methanation of the anaerobic degradation process of proteins.[5] The degradation products of these amino acids are organic compounds (short-chain and branched-chain organic acids), ammonia, carbon dioxide, and small amounts of hydrogen and sulphur compounds.[5] Amino acids are decomposed in two ways.[6] The first is deamination through a Stickland reaction, when two types of amino acids are injected. One side of the amino acid (containing the majority of the carbon atoms) acts as an electron accepter, and the other side (containing one or only a few carbon atoms) acts as an electron donor. The reaction that takes place is the deamination by bacteria ∗ Corresponding
author: Email: [email protected]
© 2013 Taylor & Francis
within the clostridium species (obligatory anaerobe).[6] It is known that these clostridium bacteria are unable to deaminate single amino acids.[7] The second type of amino acid decomposition occurs through the general fermentation process of single amino acids. Among these two types of the degradation, the fermentation of amino acids by the Stickland reaction is known to be the dominant reaction.[6] Elbeshbishy and Nakhla determined that a ratio of 8:2 (protein:carbohydrate) enables the most efficient anaerobic digestion when co-treating protein and carbohydrates.[8] Also, the amounts of organic matter removal and methanation decreased with higher protein content. In this case, it was reported that anaerobic digestion based only on protein was the least efficient. Another study reported that when protein was removed through an isoelectric point and anaerobically digested, the anaerobic efficiency increased. Also, if protein content increased, then the efficiency level decreased.[9] This result is due to the low C/N ratio, propionic acid accumulation, and hindrance of the microorganism activity due to the increase in the concentration of the ammonia-nitrogen, which was formed from proteolysis. However, Wagner and researchers stated that the methane production rate has no correlation with the carbon content in the anaerobic digestion of different kinds of protein sources and amino acids, and that the C/N ratio also had no effect.[10] It is difficult to correctly identify the methane production amount per organic compound removed since
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the latter was not taken into account when the reaction subsided. However, it is apparent that the anaerobic degradation efficiency of each amino acid that forms protein differs from those of the others. Therefore, for this study, we chose various amino acids [11,12] that are known to be created from the hydrolysis of sewage sludge and sorted them as neutral non-polar, neutral polar, alkaline, and acidic. Then, the anaerobic degradation process was evaluated in each group individually. In order to identify the degradation efficiency and the time required for the individual amino acids existing as organic nitric compounds to break down into volatile acid and ammonia, the T-N concentration of each amino acid was set at a fixed value. Also, the deamination of amino acids during the experiment increased the ammonia-nitrogen concentration, which made it possible to identify the degradation rate of amino acids. As the reaction proceeded, each item’s anaerobic resolution was identified through the change in its concentration and the methane production amount. Materials and methods Test equipment The Biochemical Methane Potential test [13] was used in this study in order to identify the anaerobic degradation process of each amino acid. For the experiment, we injected 25 mL of synthetic wastewater with amino acid and the same amount of sludge into a 125 mL serum bottle that was then placed in a thermostatic chamber at a warm temperature of 35◦ C. Before sealing the bottle, any internal oxygen was removed and plenty of nitrogen gas was injected in order to ensure that the sample was anaerobic. Nine serum bottles of the same size were made for each synthetic wastewater sample containing amino acid. A total of 90 bottles were used in the experiment including the nine control serum bottles. In order to examine the changes in the amino acids, chemical oxygen demand (COD), T-N, NH+ 4 -N, and pH during the reaction, samples were taken from each serum bottle and measured accordingly. The experimental accuracy was judged by the biogas production amount measured until two of the nine original serum bottles were left for each item. Table 1.
The gas emission measurements were taken using a manometer to measure the difference between the inner pressure of the bottle and the atmosphere. During the early part of the reaction, measurements were taken every 6 h. As the reaction time increased, measurements were taken at two and four days. The experiment was concluded when no more biogas or organic compounds remained to remove. The amount of methane produced was measured as shown below [14] VCH4 = C1 (V1 + V0 ) − C0 V0 , where VCH4 is the volume of methane produced (mL), C1 the methane content (%) at sampling time, C0 the methane content (%) at the previous sampling time, V1 the biogas volume measured by syringe (mL), and V0 the gas phase volume of the reactor (mL). Synthetic wastewater and inoculum Synthetic wastewater Synthetic wastewater, which consists of individual amino acids, is composed as given in Table 1. In order to determine the efficiency rate and time needed for each amino acid that existed in the form of organic nitrogen to decompose into volatile organic acid and ammonia (inorganic nitrogen), the synthetic wastewater was made by maintaining the T-N concentration of each amino acid at 1000 mg/L. Also, the degradation efficiency was identified through the increase in the concentration of ammonia-nitrogen caused by the deamination of the amino acid. Microelements that are known to positively affect microorganism activity was injected.[15] Microorganism seeding The microorganism that was used to identify the anaerobic degradation types of the individual amino acids was inoculated by granular sludge collected from a 35◦ C anaerobic reactor in Cheongwon-gun, Korea. The mixed liquor suspended solids (MLSS) concentration and the mixed liquor volatile suspended solids (MLVSS) concentration of the
Characteristics of each amino acid.
Name α-Alanine β-Alanine Lysine Arginine Glycine Histidine Cysteine Methionine Leucine
Molecular weight
Molecular formula
COD (mg/L)
T-N (mg/L)
NH+ 4 -N (mg/L)
pH
89 89 146 174 75 155 121 149 131
CH3 CH(NH2 )COOH NH2 CH2 CH2 COOH H2 N(CH2 )4 CH(NH2)COOH H2 NC=(NH)NH(CH2 )3 CH(NH2 )COOH CH2 (NH2 )COOH 2-NH2 -3(CH)2 N(NH)C-C2 H3 COOH HS-CH2 CH(NH2 )COOH CH3 S(CH2 )2 CH(NH2 )COOH (CH3 )2 CHCH2 CH(NH2 )COOH
6857.15 6857.15 6857.14 4428.56 3428.58 3809.53 11428.57 18285.71 17142.85
1000 1000 1000 1000 1000 1000 1000 1000 1000
0 0 0 0 0 0 0 0 0
6.93 6.99 6.86 9.39 7.03 7.46 6.58 7.02 7.04
Environmental Technology Table 2.
Characteristics of granular sludge.
Items MLSS MLVSS Soluble COD Soluble T-N Soluble NH+ 4 -N pH
Table 3. Increased T-N concentrations due to granular sludge degradation.
Concentration (mg/L) 19,900 18,500 47 21 20.25 6.93
granular sludge was 19,900 and 18,500 mg/L, respectively. Its characteristics are given in Table 2.
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Analytical methods The COD, MLSS, MLVSS, and T-N were determined according to Standard Methods.[16] The NH+ 4 -N was measured using a spectrophotometer (DR/2500, Method 8038, Nessler Method, Hach Co., USA). Only matters in a dissolved state were measured through filtering (0.45 μm) for COD, T-N, and NH+ 4 -N. A manometer was used to measure the amount of biogas production, and the methane content was measured through gas chromatography (Varian Model STAR 3400CX, USA; carrier gas, N2; injector temp, 150◦ C; column temp., 29◦ C; detector temp., 200◦ C). The pH was measured using a pH meter (Cyber scan pH 20). Results Nitrogen production caused by the hydrolysis of granular sludge T-N concentration within the serum bottle was 1000 mg/L. When 100% of the amino acid is deaminated, the ammonianitrogen concentration increases from 0 to 1000 mg/L. However, as the extracellular polymers (ECPs) from the granular sludge decomposed, the nitrogen concentration increased to a T-N concentration of over 1000 mg/L (1050– 1560 mg/L) approximately 25 days after the experiment. ECPs promote cell to be survived by protecting and maintaining the ectoenzyme of the cell surface. ECPs also act as ion-exchange resins that manage the ionic migration from the aqueous solution to the cell. Generally, ECPs are known to be primarily consisting of protein (52%), humus (41%), and carbohydrates (7%).[1] It was determined that the ammonia-nitrogen concentration reached over 1000 mg/L during the reaction because of the protein degradation (the major component of ECPs). The T-N concentration of each amino acid throughout the experiment increased more in the types of matter with low concentrations of COD injected or in those with less COD substance removal. It was because of the increase in cell lysis rate with insufficient degradable organics. It was estimated that the nitrogen increase in cysteine was higher than that the control without organic injection because a lot of cell lysis occurred when the cysteine worked as a toxicant
Increased T-N Concentration α-Alanine β-Alanine (mg/L)
50
50
Glycine 110
Histidine Cysteine 110 560
Lysine 60
Arginine 120
Methionine Leucine 240 170
for microorganisms. The increases in the nitrogen concentration for each amino acid are shown in Table 3. For clearer identification of deamination followed by the increase in the ammonia-nitrogen for each amino acid, granular sludge was injected into the control sample instead of the amino acids. Then, the control sample T-N and ammonia-nitrogen concentrations were measured according to the process of time. The results are shown in Figure 1. The increases in the T-N and ammonia-nitrogen concentrations followed a linear equation. The increase in the T-N concentration in the serum bottle was assumed to be caused by an increase in the nitrogen concentration resulting from the degradation of the ECPs. This increase is shown in Table 3. Through the increase in the T-N concentration, it was possible to calculate the ammonia-nitrogen concentration produced by the granular sludge. Then, the calculated value was subtracted from the measured ammonia-nitrogen concentration. The generation rate of the ammonia-nitrogen is given in Table 4. Ammonia-nitrogen production and COD removal Figure 2 shows the anaerobic degradation of each amino acid and the control sample (granular sludge without amino acids). The control sample had the same synthetic wastewater without the injection of any amino acids, and the increase in the nitrogen concentration caused by degradation of the ECPs was measured when no substrates remained for the microorganisms to use. As shown in (a)-(i) of Figure 2, the ammonia-nitrogen concentrations of glycine, lysine, α-alanine, histidine, and arginine increased rapidly for three days after the reaction but then began to decline. In the case of α-alanine, the concentration steadily increased until day 3 and showed a rapid increase between days 3 and 5. On the other hand, the ammonia-nitrogen concentrations of cysteine, leucine, and methionine showed steady increases with no rapid changes. Alanine is classified into α- and β-alanine according to the position of the amino groups within the molecular structure. These different structures cause different microorganism decay rates. A research by Wagner and others indicated that the individual peptone extracted from different protein ingredients shows different conversion rates under anaerobic conditions.[10] Methionine and cysteine are amino acids that deaminate through Stickland and non-Stickland reactions. Leucine
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J. Park et al. 500
500 T-N NH4+-N T-N NH4+-N
300
400
200
200
y = 9.9323x + 26.1016
100
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300
y = 12.6825x + 25.7561
0
0
5
10
15
20
T-N, mg/L
NH4+-N, mg/L
400
100
25
30
0
Day
Figure 1. Table 4. acid.
Nitrogen generation by granular sludge degradation. Generation rate of ammonia-nitrogen for each amino
NH+ 4 -N Generation (%) Glycine 91.71
α-Alanine β-Alanine 92.99 Histidine 91.34
92.99
Lysine
Arginine
91.21
94.01
Cysteine Methionine Leucine 61.55 46.61 54.59
deaminates through only the Stickland reaction.[6] The ammonia-nitrogen concentration of cysteine is higher than that of methionine due to the fact that while only C. propionicum deaminates methionine, cysteine is degraded by more types of microorganisms, which results in a higher concentration of ammonia-nitrogen. However, even though the generation rates of ammonia-nitrogen that were generated by the deamination of cysteine and methionine were 61.55% and 46.61%, respectively, no organic matter was removed. Table 4 shows the generation rates of the ammonianitrogen concentration (excluding the ammonia-nitrogen concentration from granular sludge). Although leucine is an amino acid that is known to only deaminate through the Stickland reaction,[6] this study found a deamination of 54.59% despite the limiting Stickland reaction. This result is due to the fact that as the granular sludge steadily decomposes during the reaction, it creates Stickland reaction conditions in which leucine can be decomposed. As shown in graph (j) in Figure 2, when there are no more substrates for the microorganism to use, it can be inferred that the ammonia-nitrogen concentration increased from 0 to 255 mg/L. As for cysteine and methionine, when serine exists under anaerobic conditions, it decomposes
into propionic acid and acetic acid through the Stickland reaction.[10] The deamination progress rates of these acids were 61.55% and 46.64%, respectively. Although this was an individual condition without serine, deamination was possible because the granular sludge undergoes the reaction and steadily decomposes; therefore, it cannot completely limit the Stickland reaction. Nevertheless, it is clear that the deamination of cysteine and methionine relies on the Stickland reaction, unlike the general fermentation process of the anaerobes of amino acids. Also, amino acids usually convert into volatile acids because the amino group detaches through deamination.[5] However, it was revealed that there are cases in which the organic carbon sources are not decomposed anaerobically in the case of cysteine and methionine. There could be a possibility that these amino acids could have been converted into propionate which is known as one of relatively hardly degradable volatile fatty acids. More research to clarify the deamination of cysteine and methionine is necessary. Other amino acids excluding cysteine, methionine, and leucine were not numerically recorded but showed greater than 90% ammonia-nitrogen generation and COD removal rates. Methane production according to the injected amount of COD Since the nitrogen concentration of each amino acid was fixed at 1000 mg/L, the level of injected COD was different, which made it impossible to compare the biogas production amount for each item. This is why the methane production rates per (g) COD input are calculated in Table 5. As noted in Figure 2, the methane production rates at the time of measurement were the highest between days 3
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Environmental Technology
Figure 2.
Anaerobic degradation of each amino acid.
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CH4 generation from each amino acid.
Initial COD (mg/L) CH4 generation (mL) mL CH4 /g CODin
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Initial COD (mg/L) CH4 generation (mL) mL CH4 /g CODin
α-Alanine
β-Alanine
Lysine
Arginine
6333 159.03 381.82 Glycine 3506 104.13 424.2
6333 153.81 396.09 Histidine 3767 82.69 396.36
6240 123.73 300.07 Cysteine 10,620 10.11 13.55
3041 86.27 444.7 Methionine 12,350 51.95 71.04
and 7. The α, β-alanine had a methane production as high as 68.8%. The reason that the methane production levels were maximal and then slowly declined is because that the experiment was not continuous, but a batch experiment. In the case of leucine, which has high COD input and a low COD removal rate, the methane production rate was maintained at its highest number. As with cysteine, the methane production rate appeared to be lower than that of the control bottle. Although neither methionine nor cysteine showed signs of organic matter removal from deamination half-way through the experiment, methionine showed similar levels of methane production to the control sample. This result supported that cysteine acted as a toxic substance against methanogens. Arginine, glycine, and histidine had methane production rates per COD input of 443.70, 424.20, and 396.36 mL CH4 /g CODin , respectively, and these results were higher than those for alanine and lysine. A theoretical methane production of anaerobic digestion is 395 mL CH4 /g COD.[1] The reason why the methane production rate per COD input concentration of arginine, glycine, and histidine was higher than the theoretical amount is because biogas was produced along with an increase in the nitrogen concentration when the ECPs decomposed. As mentioned earlier, the nitrogen concentration increased significantly in matter without proper COD removal or at a low COD input concentration. Likewise, the T-N concentration of lysine and alanine increased 60 and 50 mg/L, respectively, when the reaction ended. On the other hand, arginine, glycine, and histidine showed increases in 120, 110, and 110 mg/L, respectively. Therefore, it can be inferred that as high as was the increase in nitrogen concentration from the granular sludge, the related biogas production rates would have been even higher. Cysteine, methionine, and leucine had low removal rates of organic matter and showed very low methane production rates of 13.55, 71.04, and 80.77 mL CH4 /g CODin , respectively. Especially for cysteine, the methane content was maintained at approximately 7% during the experiment, which was a lower value than in the control sample. Cysteine is known to decompose into acetic acid, ammonia, carbon dioxide, hydrogen sulphide, and hydrogen through the Stickland reaction.[6] In this study, cysteine did not decompose under anaerobic conditions and was found to limit methanation activation. Therefore, whether or not
Leucine 15,833 37.41 80.77
cysteine can be decomposed under anaerobic condition needs to be assessed under mixed conditions that involve the Stickland reaction. Conclusions The results of the anaerobic degradation estimation for individual amino acids in order to estimate the anaerobic digestion of the amino acids that are formed by the hydrolysis of sewage sludge are as follows: 1. More than 90% of α-alanine, β-alanine, lysine, arginine, glycine, and histidine was converted into ammonia-nitrogen due to the deamination of amino acids, but only 61.55%, 54.59%, and 46.61% of cysteine, leucine, and methionine, respectively, were converted into ammonia-nitrogen. 2. It has been found that cysteine, leucine, and methionine, which showed low production rates of ammonianitrogen, require the Stickland reaction as a necessary condition for complete degradation. If that condition is met, more research is required as to whether the degradation product, which is produced along with the ammonia-nitrogen, can be decomposed under anaerobic conditions. 3. Although the methane production rates according to COD input of arginine, glycine, and histidine were 444.70, 424.20, and 396.36 mL CH4 /g CODin , respectively, these values are higher than the theoretically estimated value. This result is due to the fact that the degradation of the granular sludge ECPs caused gas production. In turn, cysteine, leucine, and methionine, which had low levels of ammonianitrogen production, had very little organic matter removal. This also resulted in methane production rates per COD input of 13.55, 80.77, and 71.04 mL CH4 /g CODin , respectively, which is much lower than the rates for other amino acids. This study assessed the anaerobic digestion of individual amino acids. Although not all of the existing amino acids were assessed for anaerobic digestion, this study found that amino acids other than cysteine, leucine, and methionine were easily decomposed anaerobically. As for cysteine, leucine, and methionine, an assessment is necessary under
Environmental Technology mixed conditions and involving the Stickland reaction because they are not decomposed well under individual conditions. If these three amino acids are present in high concentrations in wastewater and do not satisfy the Stickland reaction, they will cause a less efficient anaerobic digestion process. Funding This research was supported by the Energy Non-CO2 Greenhouse Gas Group. We deeply appreciate their support [Project Number: 201200000001521].
[8] [9] [10]
[11]
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