enterobacterial and viral decay experimental models for anaerobic digestion of piggery waste

Appl Microbiol Biotechnol (1992) 38:291-296 App .. Micmb/o/ogy B/otec © Springer-Verlag 1992

Enterobacterial and viral decay experimental models for anaerobic digestion of piggery waste A. Mateu ~, J. Mata-Alvarez z, and R. Par~s 1

1 Departament de Microbiologia, Facultat de Biotogia, z Departament d'Enginyeria Quimica, Facultat de Qulmica, Universitat de Barcelona, Marti i Franqu~s 1, 6p, E-08 028 Barcelona, Spain

Received 20 January 1992/Accepted 12 June 1992

Summary. A laboratory study was conducted to determine the effects of the continuous mesophilic anaerobic digestion of raw pig manure in two types of enteropathogenic microorganisms, bacterial and viral. Faecal coliforms (indigenous to pig manure) and coliphage f2 (ATCC 15766 B1) were used as a model for some indigenous enteropathogenic microorganisms. The study was completed with laboratory survival experiments in lagoon stabilization of raw pig manure, for both models. Experiments for f2 survival in cell-free synthetic medium were also carried out. The results show that the anaerobic digestion process is more effective in eliminating viral than bacterial particles. Some parameters re- lated to the ultimate biogas yield and kinetics were also determined. Lagoon stabilization of raw pig manure provides a more suitable environment for the removal of faecal coliforms than does anaerobic digestion. Finally, it was concluded that volatile fatty acids appeared to be responsible for the elimination of faecal coliforms. The agent that causes f2 inactivation is not well identified, although in some cases it could be NH3 that seems to act as a viricidal agent.

Introduction

Anaerobic digestion is a well-known technology in which organic matter is partially broken down. This process can be used for the treatment of effluents from intensive piggery farms. The advantage of this biotechnology is that organic matter is partially converted into methane gas, which may be used as a fuel and may supply part of the high energy requirements of the piggery farm (van Velsen 1981).

Effluents from intensive piggery farms may contain a large number of potentially pathogenic organisms, which can be a problem if the digested waste is applied to soil. According to several authors (Chandler and

Craven 1981; Chandler et al. 1981; Derbyshire and Brown 1978), the ability to persist in the environment is variable. Even though thermophilic anaerobic digestion produces sludges containing low levels of infectious organisms (Berg and Bergman 1980), this treatment has some problems associated with process stability and ammonia inhibition, probably due to the high tempera- tures. As a result it is not often used. Although a similar potential in eliminating pathogens during mesophilic anaerobic digestion has been referred to, this is not well known. Thus, the present laboratory study was ad- dressed to determine the most probable number (MPN) removal of indigenous faecal coliforms (FC) from raw pig manure by semicontinuous mesophilic anaerobic digestion of manure. Under the same conditions, a second goal was to determine the plaque-forming units (PFU)/ ml removal of f2 bacteriophage (ATCC 15766 B1). The f2 bacteriophage was mixed with raw manure before digestion. The experiments with the bacteriophage were possible because it was not able to multiply in raw or digested manure, as we previously determined, and, moreover, there was not any indigenous bacteriophage in raw or digested manure capable of infecting the host bacterium Eseherichia coli ATCC 15766, which was used for f2 titrations.

Technical problems of occurrence and concentration of indigenous pathogens can be avoided by using the cited microorganisms as an indicator model. The removal of indigenous FC during anaerobic digestion can be linked to the removal of enteropathogenic bacterial The removal of f2 can be linked to the removal of some indigenous enteric viruses (Bertucci et al. t977; Mathews 1971).

The present study was completed with laboratory batch experiments simulating lagoon stabilization of raw pig manure, in order to determine the survival of two models under these conditions. Similar experiments with cell-free synthetic medium were also performed for f2 bacteriophage.

Correspondence to: J. Mata-Alvarez

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Materials and methods Table 1. Coliphage f2 adsorption to particulate matter

Enumeration o f faecal coliforms. A table of the MPN, using three tubes for each dilution, was used for counting faecal coliforms (Standard Methods, APHA 1985). Samples of raw or digested manure, previously diluted in a 1/4 ringer, were tested for total coliforms by incubating at 30 ° C in lactose broth for 48 h. The po- sitive samples were then tested for faecal coliforms by incubating at 44°C in a brilliant green lactose bile broth for 24 h. Simulta- neously, the indol formation from tryptophane was determined under the same conditions (Rodier 1978).

Enumeration and growth o f f 2 bacteriophage. Viral assays were made by plaque count methods in nutrient agar plates (Adams 1959). An appropriate buffer dilution of a sample containing the bacteriophage was mixed with 3 ml of a semi-solid agar and with a drop of a concentrated suspension of the host bacterium (E. coli ATCC 15766) growing exponentially at 37°C. Then the mixture was poured over a solid agar plate. Three plates were used for each dilution. These plates were then incubated at 37°C for 5 h before counting plaques. The media used for E. coli ATCC 15766 was the nutrient broth Standard I, Merck with (g/l): special pep- tone, 15.6; yeast extract, 2.8; sodium chloride, 5.6; D( + )glucose, 1.0; pH at 37 ° C, 7.5. Solid and semi-solid agar were prepared by adding purified agar to the cited broth at concentrations of 1.5 and 0.6%, respectively.

Stock cultures of f2 were prepared incubating exponentially growing E. coli ATCC 15766 with a drop of purified bacteriophage and incubating it at 37°C for 5 h. Then the cultures were clarified by centrifugation for 20 min at 4000g. Finally the super- natant was filtered (0.22 ~t, Millipore filters). The liquid obtained, containing purified bacteriophage, was directly assayed for phage survival in synthetic medium or was mixed with raw residue for the experiments.

Experimental equipment and analysis. The experiments were conducted in three laboratory digestors, with a capacity of 21 each. They were mechanically mixed every 60 min for 2 rain at 100- 150 rpm. The reactors were placed in a 37 ° C water bath and were batch-fed daily and stirred by a mechanical device. The retention times used were 18 days for digestor 1, 14 days for digestor 2 and 10 days for digestor 3. Both the retention time and the volatile solids (VS) contents of the feed were maintained constant during the study, in order to reach the steady-state conditions of the reactors. When this condition was achieved, some physico-chemical parameters were determined in both the influents and effluents of the digestors. These were total solids (TS) and VS, pH, volatile fatty acids (VFA) and also biogas production and its methane content. Gas production was measured daily, before feeding and drawing, by a liquid displacement system, using a mixture of HC1 and distilled water, at pH 1.0, atmospheric pressure and room temperature. The measure was then corrected at 273 K and 760 mm of Hg. The methane contents of the biogas was analysed twice a month by means of gas chromatography. TS and VS, pH values and VFA in both raw and digested manure were determined on a 3 days per week basis. TS and VS were determined according to the procedure described in Standard Methods (APHA 1985). The VFA were determined directly from the aqueous sample, after centrifugation (10 min at 7500g), by gas chromatography using a 3 m × 3 mm O.D. steel column packed with 100/120 Chromosorb WAW 6.

At the same time, according to the procedure described, the survival results of two models were determined twice a week. Sam- ples coming from effluents were immediately tested for survival of coliforms and viruses. F2 bacteriophage was mixed with influent and its concentration in the feed was maintained at 10 7 PFU/ml throughout the period of study.

Additional batch experiments with raw pig manure were carried out in 250-ml glass bottles, placed in a temperature-controlled room at 37 ° C, without mixing. The pH and VFA concentration

Temper- Stock Sample Dilution Recovery ature concert- of f2 (° C) tration (PFU/ml)

of f2 (PFU/ml)

4 2.11 × 10 9 Raw manure 1 : 100 2.87 x 10 7

37 2.12×101° Rawmanure 1:100 ~1.75x108 37 2.25 x 101° Digested manure 1 : 100 1.45 x 10 s

Recovery of plaque-forming units (PFU) after mixing 1 ml of purified f2 bacteriophage stock with 100 ml of raw or digested manure for 2 h, at 37 or 4 ° C

were determined after 10, 14 and 18 days and, at the same time, the survival of indigenous FC and f2 bacteriophage was determined. The initial concentration of phage w a s 10 7 PFU/ml, the same concentration that was used for experiments with the reactors. The f2 survival batch experiments with cell-free synthetic medium (Standard 1, Merck) were carried out in culture tubes and under the same experimental conditions described for experiments with raw pig manure. The initial concentration of the bacteriophage w a s 10 9 PFU/ml.

Some experiments performed before adding the bacteriophage to the digestors, by mixing it with the feed, showed that f2 was unable to adsorb to suspended solids of raw or digested manure (Table 1), or at least, it was easily recovery from these solids at both 4°C and 370 C. Adsorption to the suspended solids of raw manure at 4°C would make difficult the homogeneous distribu- tion of phage in the digestor feed, which was stored at that temperature. Adsorption to solids of digested or raw manure at 37 ° C would make difficult the study of phage inactivation in the effluents of the digestors and also in batch experiments with raw manure respectively.

Raw pig manure was obtained from piggeries and stored at 4 ° C. It had a VS content of 21.6 g/I, a VFA content of 6.2 g/1 and a pH of 7.8.

Results and discussion

The main physico-chemical results obta ined at the three hydraul ic re ten t ion t imes (HRT) studied are presented in Table 2. The me thane content o f the biogas was about 68% in all the digestors. The p H mean value in the digestors was a round 8.0. The mean V F A value was 6200 mg/1 at the digestor inlet, whereas in the digestor eff luents it was a round 300-900 mg/1, which can be con- sidered ra ther low. The VS mean values in the eff luents o f the three digestors were very similar. The specific gas p roduc t ion (SGP), expressed as ml b iogas /g VS fed can be computed as a measure o f substrate ut i l izat ion. The results showed that this pa ramete r f luctuates a round 400 ml b iogas /g VS added. This pa ramete r can be used to evaluate two characterist ics o f the substrate: the bio- degrada t ion potent ia l and the kinetics o f this b iodegrad- at ion. Using the procedure suggested by Mata -Alva rez et al. (1990) and assuming a first order kinetic mode l for b iodegrada t ion (Mata-Alvarez and Cecchi 1990), the fo l lowing expression can be obtained:

Bo SGP = (i)

1 + 1 / ( k . H R T )

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Table 2. Experimental conditions and results obtained for batch and continuous experiments carried out with raw pig manure

Continuous experiments

Parameters Retention time (days)

10 14 18

Gas production (GP, ml/1 digested day) 847 630 525

Gas composition (% CH4 in biogas) 63.6 67.6 67.9

Volatile solids (VS, g/100 ml) 1.04 0.95 1.05 pH 8.0 7.9 8.1 Volatile fatty acids (VFA, mg/1) 620 920 290 VS removal (%) 51.8 56.0 51.4 Specific GP

(SGP, ml biogas/g VS) 392.1 4 0 8 . 3 437.5

Batch experiments

Days pH YFA

4 7.9 ND 10 7.6 3800 14 7.7 4600 18 7.7 3500 28 7.7 ND

All the results corresponding to the effluents of the digestors represent the mean value of the different measures obtained throughout the period of the study. In the experiments corresponding to raw manure, results represent the mean value of two samples (one of each bottle) coming from the same experiment. ND, not determined

where Bo is the ultimate biogas yield, that is, the biogas that could be obtained after an infinite HRT, k, the first order kinetic constant, and SGP, the specific gas production. Using a non-linear regression, a rough estimate of both parameters, Bo and k, could be obtained. The values obtained were: B 0 = 5 0 0 m l biogas/g VS fed; k = 2 . 8 days, which are in agreement with other published values (Mata-Alvarez and Cecchi 1990). Using the value of Bo, an estimation of the biodegradation achieved can be estimated. Using the actual biogas pro- duced in terms of SGP and referring it to the maximum obtainable, Bo, a percentage of the maximum biodegradation can be computed (Mata-Alvarez et al. 1990):

fB = 100" SGP/Bo (2)

The values for fs at H R T 10, 14 and 18 days are 78%, 81.6% and 87.5% respectively. As could be expected, this percentages increase with increasing hydraulic retention time used.

Results o f the survival experiments

The survival experiments of both FC and f2 bacteriophage during anaerobic digestion were carried out during the period of steady-state conditions. These results are plotted in Figs. 1 and 3, respectively. The results of both models obtained in batch experiments with raw

c )

E o

~ ' 3 ~

_0 2 _

O " 1

4-

1'0 ~0 3'0 4'0 5'0 6'0 7'0 8'0 9'0 11~0 D(:lys

Fig. 1. Survival of faecal coliforms in continuous experiments. Each point in the ordinate was obtained by calculating the mean value between two point results coming from two samples: ~, digestor at 10 hydraulic retention times (HRT); A, digestor at 14 HRT; (3, digestor at 18 HRT; ~q, feed

d

2-

1 0 ~ 1~ 1~ 2'0D~ys25

Fig. 2. Survival of faecal coliforms in batch experiments with raw pig manure. Each point corresponds to the mean value of two separate experiments carried out under the same experimental conditions

manure are plotted in Figs. 2 and 4. Figure 4 also in- cludes the data obtained in f2 survival experiments with cell-free synthetic medium.

The removal of FC was relatively high in the digestors (Fig. 1) and seems to be independent of the retention time, since the removal reached two orders of magnitude in all retention times studied. Batch experiments with lagoon stabilization of raw manure (Fig. 2), showed a higher degree of FC removal than anaerobic digestion. Lagoon stabilization of raw manure also showed a higher removal at a longer time.

The results shown in Fig. 3 indicate a high bacteriophage removal in the digestors since the removal reached four orders of magnitude at 18 days, two orders at 14 days and three orders of magnitude at 10 days. There was no clear retention time influence on the percentage phage removal.

294

8 j

7-

1 1 0 ~ ~ I t ~ I i i

20 30 40 50 80 70 80 90 Days

Fig. 3. Continuous phage survival experiments. Each point in the ordinate was obtained by calculating the mean value between two point results coming from two samples. For symbols see Fig. 1: PFU, plaque-forming units

9-

8-

(D

5-

3, 0 10 15 20 25Days20

Fig. 4. Batch phage survival experiments with synthetic medium and raw pig manure at 370 C: ©, raw pig manure, the points representing the mean value of two samples (one of each bottle), be- longing to the same experiments; E], synthetic medium, the points representing the mean value of two separate experiments carried out under the same experimental conditions

Table 3. Survival of f2 in raw manure and synthetic medium (Standard I, Merck) at 4°C

Raw manure Synthetic medium

Date Recovery of f2 Date Recovery of f2 (PFU/ml) (PFU/ml)

19/02 1.54 X 107 08/03 1.36 x 1011 01/03 2.22 x 10 v 22/03 9.10x 101° 05/03 1.76 x 107 26/03 1.28 x 1011 20/03 9.20x 106 06/04 1.10x 1011

Each group of results represents a time series of analyses from the corresponding initial sample

Anaerobic digestion, however, seems to play a sec- ondary role on phage removal, since both batch experiments at 37°C with raw pig manure and cell free synthetic medium (Standard 1, Merck) also achieved a high degree of removal (Fig. 4), although generally reaching lower levels than those obtained in the digestors. Similar batch experiments with both raw manure and synthetic medium carried out at 4°C showed no phage inactivation during the same period of time (Table 3).

Survival o f faecal coliforms

There is undoubtedly a lethal factor in the digestors causing a larger death rate than the growth rate, since the FC concentration in the effluents was lower than the concentration in the influent. On the other hand, the same mean value of elimination was observed at different retention times (Fig. 1). This behaviour can be explained by assuming the initial presence of the bactericidal agent in the digestors, which is almost completely destroyed during fhe anaerobic digestion process. This removal occurs at the same rate for all retention times. So, the contact between bacterial cells and this lethal factor is the same at different retention times and also the degree of bacterial removal. Then, only a small quantity of bactericidal agent remains in the digestors, the bacterial growth rate reaching the same mean value as the death rate. Because of that, the bacterial concentration in the effluents was invariable, whatever the retention time.

The different process operation, continuous or batch, in digestors or in lagoon stabilization of raw pig manure, respectively, could not be the only factor that in- fluences the survival results. The higher removal achieved in batch experiments with raw pig manure is probably associated with the higher level of VFA (Table 2). Moreover, these acids remained invariable and at the same concentration level at all times studied, and thus, a higher removal at a longer contact time was observed (Fig. 2) (the growth rate would be lesser than the death rate). On the other hand, it is known that VFA are rap- idly destroyed during anaerobic digestion. In our experiment, these acids had a low concentration at the retention times studied (Table 2). The association of VFA with the lethal factor would explain the difference between FC survival patterns in digestors or in batch experiments with raw manure and at the same time can be in accordance with the characteristics for the lethal factor in the digestors.

• Several authors have found an inhibitor or bactericidal fatty acids activity on a number of enterobacteria- ceae species, at low pH, in both synthetic medium (Goepfert and Hicks 1983) and in anaerobically fermented pig waste (Henry et al. 1983). Hence the present results would agree with the findings reported in the literature, in spite of the relatively high pH in our study in both environments (Table 2).

Survival o f f2 bacteriophage

Survival batch experiments of f2 at 4°C with raw pig manure and also with synthetic medium showed no phage inactivation during 1 month (Table 3). Similar experiments at 37°C showed an important removal after the same length of time (Fig. 4). Obviously, temperature plays an important role in phage removal and is probably the only factor causing phage inactivation in synthetic medium. However, regarding Fig. 4 and compar- ing the phage removal in synthetic medium and in raw manure, it seems that an additional inhibitor agent could exist in raw manure during the first half of the experiment; on the contrary, a protective factor seems to act in the second half.

Ward et al. (1976) reported the results of some stud- ies on inactivation of laboratory strains of polio viruses in laboratory experiments with millilitre quantities of raw domestic sludge, anaerobically digested sludge and also buffer solution. They claimed that a conservative factor may be present in the solids of raw or anaerobically digested sludge. Referring to the inhibitor factor, Burge et al. (1983) reported that f2 bacteriophage (ATCC 15 766 B) could be inactivated by uncharged ammonia in a buffer solution. Moreover, they found that laboratory strains of poliviruses were more quickly inactivated than f2 bacteriophage under the same conditions.

Ward and Ashley (1977) demonstrated that laboratory strains of polio viruses seeded into millilitre quantities of anaerobically digested sludge obtained from a sewage treatment plant, were inactivated by uncharged ammonia. However, this assumption could not explain the nature and characteristics of the inhibitor factor in our experiments with lagoon stabilization of raw pig manure. Effectively, there is no relationship between the slope of phage decay and free nitrogen evolution (a steady increase in NH3 concentration with time can be expected). Moreover, at the pH value of raw pig manure, most of the ammonia is in a charged state, which is probably non-toxic (Ward and Ashley 1977; Burge et al. 1983). On the other hand, it seems that the NH3 concentration could have an inhibitory effect on anaerobic digestion. Effectively there is a relationship between the pH of digestor effluent and the removal of f2: the higher the pH

295

and thus the uncharged ammonia concentration, the higher the removal achieved, since the f2 removal is greater in the digestor at 18 days retention time, which has the highest pH, and is smaller in the digestor at 14 days, which has the lowest (Fig. 3, Table 2).

It is important to point out that the fact that viral inactivation is greater in digestors than in batch experiments with raw manure (Figs. 2 and 4) would indicate that VFA are not responsible for virus inactivation, since these acids are at lower concentrations in digestors than in batch experiments with raw manure (Table 2). Moreover, the pattern of phage removal, in continuous and also in lagoon stabilization of raw manure, would probably be different if VFA were the lethal factor.

Comparison of results with others in the literature

From the literature, the reduction of viruses under laboratory conditions with other substrates appears to be a little bit higher than the reduction found here. Table 4 summarizes some of the results published on the topic. The reasons for the differences can be discussed not only in terms of the scale or nature of the digestion process (continuous or batch), or even in terms of the different types of sewage, possible pretreatments before anaerobic digestion or type of viruses (vaccinal or wild strains), but can also be explained by considering the method used in the addition of viruses to the anaerobically digested sludge. It seems that lower inactivation will be achieved if the viruses are added to the digestors by mixing them with the feed of the digestors, as previously mentioned by Sanders et al. (1979). The removal results presented here lie approximately between those obtained in the full-scale experiments and those obtained by the cited authors in the laboratory.

Comparison between survival results obtained for both models

As determined by the present conditions, the rate of inactivation of f2 bacteriophage during mesophilic anaerobic digestion is greater than the rate of inactivation of FC. These results contradict the data obtained by other

Table 4. Some survival experimental results of other authors in the literature

Factor: Enteroviruses coil- phage MS2

Medium: Urban sludge

System: CSTR Temperature (° C): 35 Time (h): 24 Average removal (%): 54.4-97.6 Reference: Bertucci et al. (1977)

Free Polio viruses

Pig dung, Rice straw, Human excreta Batch system 35 24 96.8 Yah-We-an et al. (1985)

Indigenous viruses

Urban sewage sludge

CSTR 30 35 days 88 Goddard et al. (1981)

Indigenous viruses

Urban sewage sludge

CSTR 35 21 days 90 Berg and Bergman (1980)

CSTR: continuous stirred tank reactor

296

au thors , who worked with o ther k inds o f substra tes . F o r ins tance Berg and Bergman (1980) f o u n d a 90% remova l o f ind igenous viruses and a 98% remova l o f ind igenous F C dur ing domes t ic raw sludge anae rob ic d iges t ion in a sewage t r ea tmen t p lan t by a con t inuous p rocedure at 20 days re ten t ion t ime at 37 ° C.

Conclusions

The po ten t i a l o f semicon t inuous anae rob ic d iges t ion o f raw pig manure in e l iminat ing these types o f mic roo r - ganisms has been demons t r a t ed for bo th vi ra l and FC. The remova l ra te ranged be tween 97 and 99%. The results show a d i sagreement be tween bac te r ia l and viral behaviour . I t appears tha t the ra te o f r emova l is greater for viral par t ic les than for bac te r ia l cells for anae rob ic digest ion, assuming tha t viral par t ic les do no t mul t ip ly , whereas bac te r ia l cells can mul t ip ly under the present condi t ions . Unl ike the f2 results , l agoon s tab i l iza t ion o f raw pig m a n u r e seems to be more sui table for F C remova l than anae rob ic digest ion. V F A seems to p lay an i m p o r t a n t role in the inac t iva t ion o f FC. Inac t iva t ion o f co l iphage f2 seems to be l inked to the presence o f free a m m o n i a . However , more exper iments are needed to subs tan t ia te this hypothes is .

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enterobacterial and viral decay experimental models for anaerobic digestion of piggery waste

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