Jul 18, 1994 - Springer-Verlag 1995. B. K. Ahring ⢠M. Sandberg ⢠I. Angelidaki. Volatile fatty acids as indicators of process imbalance in anaerobic digestors.
Appl Microbiol Biotechnol (1995) 43:559-565
© Springer-Verlag 1995
B. K. Ahring • M. Sandberg • I. Angelidaki
Volatile fatty acids as indicators of process imbalance in anaerobic digestors
Received: 18 July 1994/Received revision: 5 October 1994/Accepted: 10 October 1994
Abstract In continuously stirred tank reactor experiments, with manure as substrate at thermophilic temperatures, the use of volatile fatty acids (VFA) as process indicators was investigated. Changes in ¥ F A level were shown to be a good parameter for indicating process instability. The VFA were evaluated according to their relative changes caused by changes in hydraulic loading, organic loading or temperature. Butyrate and isobutyrate together were found to be particularly good indicators. Butyrate and isobutyrate concentrations increased significantly 1 or 2 days after the imposed perturbation, which makes these acids suitable for process monitoring and important for process control of the anaerobic biological system. In addition it was shown in a batch experiment that VFA at concentrations up to 50 mM did not reduce the overall methane production rate. This showed that ¥ F A accumulation in anaerobic reactors was the result of process imbalance, not the cause of inhibition, thus justifying the use of VFA as process indicators.
Introduction With the increasing full-scale application of anaerobic digestion for waste treatment and biogas production there is a need to develop reliable methods for the evaluation and control of the anaerobic digestion process. Monitoring of the anaerobic process requires
B. K. Ahring ( ~ ) - M. Sandberg The Anaerobic Microbiology/Biotechnology Research Group, Department of Environmental Science and Engineering, Building 115, The Technical University of Denmark, 2800 Lyngby, Denmark I. Angelidaki Department of Biotechnology, Building 223, The Technical University of Denmark, 2800 Lyngby, Denmark
access to a suitable parameter reflecting the metabolic state of the process. Anaerobic digestion is a complex process consisting of a series of microbial reactions catalyzed by consortia of different bacteria (McInerney et al. 1980). The interdependence of the bacteria is a key factor of the biogas process. Under conditions of unstable operation, intermediates such as volatile fatty acids (VFA) and alcohols accumulate (Gujer and Zehnder 1983) at different rates depending on the substrate and the type of perturbation causing instability (Allison 1978). The most common disturbances causing imbalance are hydraulic or organic overloading, the presence of inorganic or organic toxins or other disturbances in the process conditions such as temperature and substrate changes (Switzenbaum et al. 1990). Several parameters have been suggested as stress indicators. Some of the most commonly used indicators include measurements of gas production, gas composition, pH, volatile solids destruction and VFA concentrations. In general, most of these indicators are suitable for detecting gradual changes. However, pH, volatile solids reduction and gas composition are often too slow for the optimal detection of sudden changes (Angelidaki and Ahring 1994). pH changes are small in highly buffered systems, as often seen in reactors with high ammonia loads, even when the process is severely stressed (Angelidaki and Ahring 1994). Some important features of a good process indicator are its ability to detect imbalance at an early stage and its ability to reflect the metabolic state of the system directly. It is also important that the relative change of the parameter following a perturbation is significant compared to background fluctuations and analysis uncertainties. For a long time it has been recognized that the VFA concentration is one of the most important parameters for the accurate control of anaerobic digestion (Chynoweth and Mah 1971; Fischer et al. 1983; Hill and Bolte 1989; McCarty and McKinney 1961).
560
Volatile fatty acid accumulation reflects a kinetic uncoupling between acid producers and consumers and is typical for stress situations. Many investigators have correlated the process stability to the concentrations of individual VFA in the reactor (Hill et al. 1987; Kaspar and Wuhrmann 1978; Hill and Holmberg 1988; Varel et al. 1977). Acetate concentrations higher than 13 mM have been suggested to indicate imbalance (Hill et al. 1987). Propionate has been suggested by some investigators to be a better indicator of process instability (Kaspar and Wuhrmann 1978; Varel et al. 1977). Hill (1982) proposed that the propionate/acetate ratio should be used as a process indicator. He suggested that for a stable process the propionate/acetate ratio should be below 1.4. Longer-chained volatile fatty acids (C4-C6), and especially their isoforms, have also been suggested as process indicators (Fischer et al. 1983; Chen and Day 1986). Hill and Holmberg (1988) showed that isobutyrate or isovalerate concentrations below 0.06 mM indicate a stable process, while concentrations between 0.06 mM and 0.17 mM are a signal of process imbalance. However, from the many different levels of VFA found in different reactor systems, it can be concluded that it is not feasible to define an absolute VFA level indicating the state of the process. Different anaerobic systems have their own "normal" levels of VFA, determined by the composition of the substrates digested or by the operating conditions (Angelidaki et al. 1993). The toxic effects of high VFA concentrations on the anaerobic digestion process have been studied and reported by several authors (Ahring and Westermann 1988; Gorris et al. 1989; Gourdon and Vermande 1987), and the resulting drop in pH is generally considered to be the main cause of the toxicity (Hill 1982; Mosey and Fernandes 1984). Several studies have shown that high concentrations of VFA in themselves have no adverse effect on the biogas process (Boone 1980; Gourdon and Vermande 1987). However, it has been shown that degradation of propionate and butyrate is inhibited by acetate (Ahring and Westermann 1988; Kasper and Wuhrmann 1978). The specific role of the individual VFA in the overall anaerobic process has been the subject of debate and is still not completely understood.
Table 1 Operation conditions after perturbation in the continuously stirred tank reactor experiments (VS volatile solids, HRT hydraulic retention time, T temperature, LR loading rate)
In the present study the VFA were evaluated as process indicators by imposing different types of perturbation imbalances in experiments with continuously. stirred tank reactors fed with manure under thermophilic temperatures. In addition, the effect of VFA on methane production rates during the digestion of manure was examined in batch experiments.
Materials and methods Continuously stirred tank reactor experiments The experiments were performed in four 3-1, automated laboratoryscale continuously stirred tank reactors with a working volume of 21. The reactions were automatically fed at intervals of 6 h from a substrate storage bottle stirred for 10 min before pumping of the feed. The reactors were inoculated (100% inoculum) from a full-scale reactor operating with the same type of substrate. The temperature and hydraulic retention time were chosen as 55°C and 15 days respectively, corresponding to the operation conditions of several new full-scale biogas plants in Denmark.
Substrate Manure (75% cattle and 25% swine) obtained from two different Danish biogas plants was used as substrate. The two batches were mixed resulting in total solid and volatile solid concentrations of 5.1% and 4.1% respectively. The total ammonia concentration was 2.1 g N/1.
Experimental design Two series of experiments were conducted, where the response to several types of perturbations was tested. Prior to initiation of each experiment all four reactors were operated for a period of approximately 1 month at steady state at 15 days hydraulic retention time, corresponding to a loading rate of 2.7 g volatile solids 1-1 day- ~. At day 0 (Figs. 1-3), perturbations were introduced by altering operating conditions such as the hydraulic reaction time, the organic load or the process temperature. The experimental design is given in Table 1.
Experiment 1. For the first reactor (RI) the hydraulic reaction time was decreased from 15 to 10 days. For the second reactor (R2) the organic content of the substrate was increased from 4.1% to 6.3%
Parameter
Reactor
Expt.
VS (g/i)
HRT (days)
T (°C)
LR (g vs 1-~ day -1)
Hydraulic overload Organic overload Temperature change Contol
R1 R~ R; Rz R3 R3 R4
1 2 1 2 1 2
41 41 63 93 41 41 41
10 5 15 15 15 15 15
55 55 55 55 51 59 55
4.1 8.2 4.2 6.2 2.7 2.7 2.7
561 volatile solids by the addition of whey. Whey was chosen because this type of waste product from diaries is often added in large batches in full-scale biogas plants in Denmark, typically causing temporary organic over-loading. To the third reactor (R3) the temperature was decreased from 55 °C to 51 °C, simulating the loss of temperature control. After the perturbations had been imposed these conditions were maintained for 1 month.
from the first day following the imposed perturbation in the other reactors to the last day of each experiment, was calculated.
2. For R1 the hydraulic retention time was decreased from 15 to 5 days, for R2 the organic content was increased from 4.1% to 9.3% volatile solids by the addition of whey and for R3 the temperature was increased from 55 °C to 59 °C. Perturbations were maintained for 10 days. For both series of experiments the fourth reaction (R4) served as a control, operated without changes during the experiments.
C o n t i n u o u s l y stirred t a n k reactor
Experiment
Batch experiments The effect of high concentrations of VFA on methane production from manure was tested in batch experiments. The experiments were conducted in l16-ml vials with 20 ml digested manure from the control reactor (R4). VFA tested were acetate, propionate, butyrate, and valerate and they were added from stock solutions as sodium salts. The methane production in the headspace of the vials was followed during the experiment. The methane production rate was calculated as the slope of the linear part of the methane-production time curve.
Analytical methods Volatile solids, total solids and pH were determined using standard methods (Clesceri et al. 1985). The CH4 and CO2 contents of the biogas produced from reactors in the continuous experiments were determined with gas chromatography using thermal conductivity detection. VFA were analyzed on a gas chromatograph equipped with flame ionization detector. The ammonia content was determined using the Kjeldahl method.
Statistical significance test To evaluate the different VFA concentrations, propionate/acetate ratio and the gas yield, as possible indicators of process imbalance, a significance value (z) of the changes after the perturbation compared to the level of the examined parameter prior to the perturbation was calculated. The z values were calculated for days 1 and 2 after the perturbation as: x-y z -
SD
(1)
where x is the measured value of the parameter at day 1 or 2, y is the average of the parameter values up to the time of perturbation, and SD is the standard deviation of the parameter value up to the time of perturbation. The average and standard deviation up to the time of perturbation include 6-18 values measured during a period of 20 days. The test was also applied to the total VFA concentration (including acetate), which was the sum of the molar concentrations of the individual VFA. In addition, the test was applied to the combined butyrate plus isobutyrate concentration, after the concentrations of each acid had been normalized with the average concentration of the corresponding acid up to the time of perturbation. The maximum z value for the control reactor, defined as the highest daily value
Results
Altering the hydraulic retention time f r o m 15 to 10 days was followed b y a decrease in the m e t h a n e p r o d u c t i o n rate a n d yield on the first day. However, the steady-state m e t h a n e p r o d u c t i o n rate increased f r o m a p p r o x i m a t e l y 0.7 to 1.0 1 (1 reactor v o l u m e ) - 1 d a y - 1. T h e m e t h a n e yield stabilized at 0.23 1/g volatile solids, which was slightly lower t h a n before the disturbance (0.27 1/g volatile solids; Fig. la). The level of all V F A increased after the p e r t u r b a t i o n (Fig. lb, c) with acetate increasing f r o m 10 m M to 28 m M 2 days after the pert u r b a t i o n (Fig. lb). The increase in the p r o p i o n a t e concentration was also significant; from a p p r o x i m a t e l y 5 m M to 25 m M over the next 7 days (Fig. lb). T h e p r o p i o n a t e / a c e t a t e ratio increased slowly a n d b e c a m e greater t h a n one only after a p p r o x i m a t e l y 10 days (Fig. lb). The increase in the concentrations of the higher V F A was significant, especially for isobutyrate, which increased f r o m a p p r o x i m a t e l y 0.13 m M to 1.0 m M in 1 day, while b u t y r a t e a n d valerate increased significantly at day 2 (Fig. lc, d). Increasing the organic content of the feed from 4.1 to 6.3 % was followed b y a gradual increase in the methane p r o d u c t i o n rate from 0.6 to 1.4 11-1 d a y - 1 . The m e t h a n e yield decreased during the first 3 days f r o m 0.25 to 0.23 1/g volatile solids, whereafter its increased to 0.35 1/g volatile solids owing to the higher gas potential of the e x t r a organic material a d d e d c o m p a r e d to m a n u r e (Fig. 2a). T h e concentrations of all the V F A measured, except valerate, increased significantly 2 days after the p e r t u r b a t i o n (Fig. 2b-d). T h e p r o p i o n ate/acetate ratio b e c a m e higher than unity after 7 days. Increasing the t e m p e r a t u r e f r o m 55 °C to 59 °C h a d a p r o f o u n d effect on all the m e a s u r e d parameters. Elevation of the t e m p e r a t u r e was followed by an i m m e d i a t e decrease in b o t h the m e t h a n e p r o d u c t i o n rate a n d the m e t h a n e yield (Fig. 3a). The m e t h a n e p r o d u c t i o n rate decreased from a p p r o x i m a t e l y 0.7 to 0.3 11-1 d a y - 1 in 1 day, while the m e t h a n e yield decreased f r o m 0.25 to 0.10 1/g volatile solids. After the p e r t u r b a t i o n all the V F A m e a s u r e d increased to high concentrations (Fig. 3b-d). T h e increase b e c a m e significant for all V F A 1 or 2 days after the disturbance, except p r o p i o n ate and butyrate. The p r o p i o n a t e / a c e t a t e ratio for this reactor was unfortunately higher t h a n 1 before the p e r t u r b a t i o n was i n t r o d u c e d (Fig. 3b); after the pert u r b a t i o n the ratio decreased f r o m a p p r o x i m a t e l y 2 to 0.3. O n l y three runs out of six, one f r o m each type of imbalance, are graphically presented (Figs. 1-3). The
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Fig. l a - d Effects produced by changing the hydraulic retention time from 15 to 10 days in the continuously stirred tank reactor experiments, a • Methane production rate, [] methane yield; b A acetate, • propionate, • propionate/acetate ratio; e V isobutyrate, • butyrate; d ~ isovalerate, i , valerate (HRT hydraulic retention time, P/A propionate/acetate ratio, d days)
results from all six runs are presented as the calculated z values in Table 2. The calculated z values that are significant within a 5% confidence interval (z > 1.96) for days 1 and 2 are given in Table 2. During the experiment the maximum z value of the control reactors exceeded 2 on a few occasions, but only twice did it reach 3.0 and 3.5, for propionate and for the propionate/acetate ratio respectively. In order to minimize detection sensitivity a higher z value could be chosen. Some of the parameters measured showed a significant change 1 day after the perturbation. All para-
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20
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Fig. 2a-d Effects produced by increasing the organic content from 4.1% to 6.3% volatile solids (VS) in the continuously stirred tank reactor from experiments. Symbols as in Fig. 1
meters, except valerate and the propionate/acetate ratio, showed a significant difference 2 days after the perturbations. The changes in methane yield were in all cases significant, except on day 1 when the temperature was decreased from 55 °C to 51 °C, with z values of 3-6. Acetate had higher z values, but failed to show significant changes at day 2 when the temperature was decreased: propionate in general had smaller z values. Changes in isobutyrate concentrations were not significant for the first 2 days after the temperature had been increased from 55 °C to 59 °C, but the isobutyrate concentration reflected process imbalance in all other cases. Butyrate concentrations increased to very high
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The methane production rate increased with increasing concentrations of VFA up to 50 m M for all VFA tested (Fig. 4). A slight decrease was observed in the methane production rate at 200 m M acetate or butyrate, while methane production from propionate decreased with the concentration of propionate or valerate was 100 mM.
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Fig. 3a-d Effects produced by changing the temperature from 55 °C to 59 °C in tile continuously stirred tank reactor experiments. Symbols as in Fig. 1
levels, with z values ranging from 3 to 54. In all cases butyrate was able to detect imbalance at day 2. Isovalerate was also able to detect imbalance in all cases at day 2 but with lower z values, ranging from 3 to 8. The propionate/acetate ratio was a very slow parameter and was not useful in detecting imbalance within 2 days. The total VFA concentration was also a good process indicator, detecting perturbations at day 2 in all cases except for temperature increases. The combined butyrate plus isobutyrate parameter detected imbal-
Discussion The significance study showed that VFA was generally a good parameter for predicting process instability and measurements of isobutyrate and butyrate were especially useful. The concentrations of most VFA changed significantly within 2 days after a perturbation was imposed. The methane yield reflected imbalance within 2 days in all the situations examined. However, the significance level of the changes was relatively small and, in some cases, the yield recovered after a few days while the VFA remained high, making an evaluation of the process based only on measurements of the methane yield doubtful. In addition, the methane yield parameter required a precise estimation of the volatile solids of the feed, which makes this parameter difficult to use for the routine regulation of biogas with varying input to the reactor. Measurements of the methane production rate were not evaluated as possible parameters, since changes could reflect the actual loading of the reactor and not only the state of the process. Of the individual VFA, butyrate and isobutyrate provided the most significant changes in most cases. Butyrate concentrations increased up to 875% within 2 days after a perturbation, and in many cases a significant increase was observed on the first day. Isovalerate could, in all cases, detect imbalance at the 2nd day, but the significance level of the changes was not as high as for butyrate. It has previously been reported that the isoforms of butyrate and valerate are the best indicators of process instability (Hill and Bolte 1989; Hill and Holmberg 1988). In our investigation, however, we found that a combination of the "normally" occurring VFA, butyrate, with its isoform, isobutyrate, provided the best indicator of process stress. A process parameter consisting of the combined concentrations of butyrate and isobutyrate relies upon the interrelation of these two isomers of butyrate. Isomerization of the acids occurs during anaerobic digestion and has often been reported in the literature (Lovley and Klug 1982; Tholozan et al. 1988; Wu et al. 1993; Stieb and Schink
564 Table 2 Significance test values (z), at day 1 and day 2 after perturbation, calculated from Eq. 1. All the values are significant at a level of 5%
(ND not determined, VFA volatile fatty acids, and P/A propionate/acetate ratio, iB + B isobutyrate plus butyrate) Perturbation
CH4 yield
Acetate 12
Propionate
Isobutyrate
12
12
Butyrate 12
Isovalerate 12
Valerate 12
P/A
VFA"
iB + B b
1 2 1 2 1 2
12 H R T 1 5 ~ 1 0 days 6 HRT15~5days 6 VS4.1%~6.3% ND VS4.1%~9.2% 2 T55°C--,51°C T55°C--+59°C 4
4 6 5 2 9 3 7 6 12 13 4 ND 6 4
2 2 ND
7 3 3 3 2
12 11 3 8 3 2 4 N D 10
9 18 54 5 18 30 ND 3 3 11
3 ND -
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3
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2 4 ND 4
6
ND -
8 7 11 5 14 16 46 2 6 4 8 10 11 18 ND 4 ND 6 2 7
a Total concentration of the measured VFA (including acetate) b Z values calculated from the sum of the normalized isobutyrate and butyrate concentrations
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150
200
Concentration (mM)
Fig. 4 Effect of various volatile fatty acids on the overall methane production rate. (Acet. acetate, But. butyrate, VaI. valerate, Prop. propionate)
1989), which justifies the combination of these two acids into one parameter. From the significance analysis it was obvious that the propionate/acetate ratio was useless in determining process instability. Of all the perturbations examined, increasing the temperature had the greatest effect on the final product of the process, i.e. methane production. Methane production almost ceased after the increase of temperature and had not resumed even 10 days later, indicating the importance of a stable temperature for the performance of the process. The individual VFA did not inhibit the overall biogas process at concentrations up to 50 mM. A slight decrease in methane production rate was observed when propionate or valerate was present at a concentration of 100 mM, while the other VFA examined did not reduce the methanogenic activity at this concentration. Addition of 200 m M VFA decreased the methanogenic activity. Therefore, VFA accumulation in an anaerobic reactor, where the concentrations of the individual VFA usually are below 50 mM, may be con-
sidered mainly as a warning and not as a cause of imbalance. The results justify the use of individual VFA as a process performance indicator. The concentrations of the individual VFA after perturbation did not reach inhibitory levels, therefore, it should be stressed that it is the relative changes of the VFA that are used as indicators for process perturbation, not the absolute concentrations. A combined parameter reflecting the concentrations of both butyrate and isobutyrate could prove a reliable tool for the early detection of stress within the anaerobic digestion process, allowing operation adjustments and thus avoiding failure of the process. Acknowledgements This work was supported by grants from the Danish Energy Council (1383/90-0001) and the Nordic Ministerial Council.
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McCarty PL, McKinney RE (1961) Volatile acid toxicity in anaerobic digestion. J Water Control Fed 33:223-232 McInerney MJ, Bryant MP, Stafford DA (1980) Metabolic stages and energetics of microbial anaerobic digestion. In: Stafford DA, Wheatley BI, Hudges D E (eds) Anaerobic digestion. Applied Science, London, pp 91-98 Mosey FE, Fernandes XA (1984) Mathematical modelling of methanogenesis in sewage sludge digestion. Microbiol Methods Environ Biotechnol 159-169 Stieb M, Schink B (1989) Anaerobic degradation of isobutyrate by methanogenic enrichment cultures and by a Desulfococcus multivorans strain. Arch Microbiol 151:126-132 Switzenbaum MS, Giraldo-Gomez E, Hickey RF (1990) Monitoring of the anaerobic methane fermentation process. Enzyme Microb Technol 12:722-730 Tholozan JL, Samain E, Grivet JP (1988) Isomerization between n-butyrate and isobutyrate in enrichment cultures. FEMS Microbiol Ecol 53:187-191 Varel VH, Isaacson HR, Bryant MP (1977) Thermophilic methane production from cattle waste. Appl Environ Microbiol 33:298-307 Wu WM, Thiele JH, Jain MK, Zeikus JG (1993) Metabolic properties and kinetics of methanogenic granules. Appl Microbiol Biotechnol 39:804-811
