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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bioreactor and reaction conditions
  6. Composition of starch mineral medium
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Thermophilic aerobic digestion(TAD), or liquid composting, is a versatile new process for the treatment and stabilization of high strength wastes of liquid or, perhaps more importantly, slurry consistency.

The pattern of inactivation of various pathogenic and indicator organisms was studied using batch digestions under conditions that may be expected to be found in full-scale TAD processes. Rapid inactivation of test populations occurred within the first 10 min from the start of digestion. The inactivation rate was slightly lower when digestions were conducted below 60 °C. In some instances, a ‘tail’ was apparent, possibly indicating the survival of relatively resistant sub-populations particularly in the case of Serratia marcescens and Enterococcus faecalis, or of clumping or attachment of cells to particulate materials. The effect of pH on the inactivation of the test populations depended on the temperature of digestion, but varied with the test population. At 55 °C Escherichia coli was more sensitive to temperature effects at pH 7 than at pH 8, but was more sensitive at pH 8, 60 °C. The reverse was the case at 60 °C for Ent. faecalis. An increase in the solid content of the digesting waste caused a progressive increase in the protection of test organisms from thermal inactivation. Challenging a TAD process with test strains allows (via estimation of D-values) a quantification of the cidal effects of such processes, with a view to manipulating process variables to enhance such effects.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bioreactor and reaction conditions
  6. Composition of starch mineral medium
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Wastes can no longer be considered as that which society needs to eliminate, as the drive for recycling of the abundant organic matter in wastes increases. However, the recycling of various waste biomasses, particularly those resulting from agriculture and animal production practices, as well as sewage, is strictly tied to their microbiological and sanitary quality. This is necessary to safeguard against the spread of disease-bearing pathogens (De Bertoldi et al. 1988). Conventional waste treatment processes, including aerobic and anaerobic digestion, have been found to be inefficient from a hygienic and epidemiological point of view, being generally incapable of achieving acceptable levels of pathogen reduction and vector attraction limit in treated wastes, namely the class A sludge quality according to the US Environmental Protection Agency (EPA) classification (De Bertoldi et al. 1988; EPA 1990, 1992; Juris et al. 1992, 1993; Plachy et al. 1993; Pagilla et al. 1996).

Limitation of pathogen levels, particularly those of human or animal origin, in wastes intended for land or sea disposal is the subject of legislation in Europe and North America (EPA 1992; Droffner & Brinton 1995) and is a major driving force for the development of aerobic thermophilic digestion. Auto-thermal thermophilic aerobic digestion (ATAD or TAD) utilizes the heat of microbial aerobic metabolism to raise the temperature of waste undergoing treatment in an insulated system to thermophilic levels (≥45 °C). Waste pasteurization results from the thermal inactivation of mesophilic, pathogenic non-spore-forming bacteria, eukaryotes and parasite eggs in the digesting waste (Kabrick & Jewell 1982). Wastes treated by this process under appropriate thermal and time conditions often meet the class A requirement of the EPA. Hence, it is currently recommended that wastes intended for use in grazing grounds, as manure or soil conditioner, or in recreational grounds be treated by this method (EPA 1992). It is also particularly suitable for wastes being considered for upgrading and recycling as animal feed supplements, since many agricultural and food industry wastes are currently employed in this capacity (Couillard et al. 1989; Hammoumi et al. 1998; Barrington & Cap 1990; Krishna & Chandrasekaran 1995).

Inactivation of waste-associated pathogens has been studied in aerobic mesophilic, anaerobic mesophilic and thermophilic composting processes (Abdul & Lloyd 1985; Olsen & Larsen 1987; Carrington et al. 1991; Kearney et al. 1993a,b; Gerba et al. 1995). However, due to the relative novelty of aerobic thermophilic digestion, few studies exist that describe the behaviour of pathogens and indicator bacteria in such a process (Kelly et al. 1993). Consequently, projections have been made on the capacity of TAD to achieve pathogen destruction based on the known sensitivity and behaviour of mesophilic indicator populations subjected to thermophilic temperatures (Messenger et al. 1993; Ponti et al. 1995a,b). This approach tends to overlook the complexity of the milieu likely to be obtained in a waste treatment of a slurry nature and the effect of different process conditions on the behaviour of the target populations.

Recent studies have shown that, in spite of the projected rapid destruction of mesophilic populations at thermophilic temperatures which prevail during composting, mesophiles can survive for long periods at temperatures higher than those likely to be employed in ATAD (Droffner & Brinton 1995).

The physico-chemical parameter which appears to have the greatest influence on the inactivation of pathogens in thermophilic aerobic digestion is temperature. The temperature–time combination has been used as the principal regulatory parameter in many processes. However, pathogens can be expected to respond variably to the effect of heat combined with different pH, dissolved oxygen and waste solid levels (Plachy et al. 1995).

As the emphasis on the hygienic quality of wastes increases, it is prudent to examine the thermal inactivation characteristics of different organisms in thermophilic biological waste treatment.

This investigation was undertaken to study the inactivation of various pathogenic, potentially pathogenic and indicator bacteria in a thermophilic aerobic process under controlled conditions of pH, aeration (dissolved oxygen) and solids content that may be expected to be found in a typical waste treatment process employing batch TAD or liquid composting.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bioreactor and reaction conditions
  6. Composition of starch mineral medium
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Test bacteria for inactivation studies

Six pathogenic, potentially pathogenic and indicator organisms were selected for the studies, Escherichia coli (NCTC 9001), Serratia marcescens (NCTC 1377), Salmonella enteritidis (NCTC 8515), Enterococcus faecalis, Pseudomonas aeruginosa (a clinical isolate obtained from Manchester Royal Infirmary, UK) and Campylobacter jejuni (NCTC 11351). Cultures were maintained in the culture collection of the Department of Bioscience and Biotechnology, University of Strathclyde.

Bioreactor and reaction conditions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bioreactor and reaction conditions
  6. Composition of starch mineral medium
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Inactivation studies were carried out in batch reactions at different temperatures and pH values in a 1·5 l (working volume), SGI 2·0 l bench fermenter (Setric Genie Industriel, Toulouse, France). Studies were carried out in a model simulated waste (starch mineral medium). The simulated waste was inoculated and digested with a combination of four thermophiles previously isolated as predominant thermophilic populations from potato peel waste undergoing aerobic thermophilic digestion. The thermophiles were tentatively identified as Bacillus spp. based on their morphology, Gram reaction and presence of endospores.

Simulated waste (1·4 l) in the bench bioreactor fitted with pH, dissolved oxygen (DO) and temperature probes, was sterilized by autoclaving at 121 °C for 15 min. The fermenter was set up at the appropriate temperature and, after calibration of the DO probe, the medium was inoculated with a cocktail of 100 ml of 24 h inoculum made up of 25 ml each of four thermophiles grown in the same medium as the simulated waste. The thermophiles were grown in shake flask at 55, 60 or 65 °C as appropriate. Inactivation studies were carried out at pH 7 or 8.

Regulation of pH was carried out automatically using 1 mol l−1 H2SO4 and 2 n NaOH. The aeration rate in the fermenter was set at 1·0 vvm (volume per volume per minute) and the agitation rate at 350 rev min−1. Pasteurization studies (inoculation of test pathogen) were started at the point where the DO tension of the digesting mass fell between 0 and 10% saturation, representing the DO level that can be expected in full scale TAD. Volatile fatty acids, known to assist pathogen destruction during waste pasteurization, can also be expected to be highest at this level of DO. Control pasteurization reactions were performed at 55 and 60 °C in 0·1 mol l−1 sodium phosphate buffer at pH 7·0, using E. coli. Sterile model waste set at 60 °C, pH 7, was used to check the effect of growing thermophiles on inactivation of E. coli.

Composition of starch mineral medium

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bioreactor and reaction conditions
  6. Composition of starch mineral medium
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

The simulated starch waste was composed as follows: soluble starch, 2% (w/v); yeast extract, 0·2% (w/v); mineral solution, 10 ml l−1; vitamin solution, 10 ml l−1 (sterilized by filtration using a 0·2 μm syringe-mounted filter (Whatman, Maidstone, Kent, UK) and added after the medium was autoclaved and cooled to room temperature); trace metals solution, 2 ml l−1. The mineral, vitamin and trace metal solutions were modified from those described by Tanner (1997). The mineral solution contained (g l−1): NaCl, 40; (NH4)2SO4, 132; KCl, 10; KH2PO4, 10; MgSO4.7H2O, 20; CaCl2.2H2O, 4 stored at room temperature until use. The vitamin solution contained (mg l−1): pyridoxine HCl, 10; thiamin HCl, 5; riboflavin, 5; calcium panthotenate, 5; para-amino benzoic acid, 5; nicotinic acid, 5; biotin, 2 stored at 4 °C until use. The trace element solution contained (g l−1): MnSO4.H2O, 1·0; Fe(NH4)2(SO4)2.6H2O, 0·8; CoCl2.2H2O, 0·02; ZnSO4.7H2O, 0·02; Na2MoO4.2H2O, 0·02.

The pH of the medium was adjusted to 6·8 before sterilization, using 1 n NaOH.

Production and inoculation of test organisms

Escherichia coli, Ent. faecalis, Salm. enteritidis, Ser. marcescens and Ps. aeruginosa were grown in tryptone soya broth at 35 °C, 200 rev min−1, in a Gallenkamp orbital shaker (Sanyo Gallenkamp plc, Loughborough, UK) for 12–18 h. Cells were harvested in late log phase (deceleration phase) by centrifugation at 12 500 g and 4 °C in an MSE high-speed 18 centrifuge (Sanyo Gallenkamp plc, Loughborough, UK) for 10 min. Harvested cells were then re-suspended in sterile saline and used to inoculate digesting waste, to a level predetermined by total viable counting to give approximately 107cfu ml−1 of waste.

Campylobacter jejuni was grown in Bacto Brucella broth (Difco, Beckton Dickenson, Oxford, UK), incubated at 42 °C in an atmosphere produced with a gas-generating kit (Campylobacter system BR060 A; Oxoid, Basingstoke, UK), for 36 h. It was harvested by centrifugation at 16 000 g and 4 °C for 10 min and used to inoculate digesting waste as above. Inoculation was carried out by rapidly pumping a suspension of the test organism into the reactor through a short length of sterile silicone tubing using a peristaltic pump.

Sampling and enumeration

Samples were collected at 5 min intervals (except for Camp. jejuni, considered to be very fragile and so sampled at shorter intervals), for the first 30 min and at 30 min intervals for up to 2 h, thereafter at 6 hourly intervals up to 24 h. Campylobacter jejuni samples were taken at 2 min for the first 10 min, and at 5 min intervals for a further 20 min thereafter then as for the other test pathogens up to a maximum of 2 h.

The first sample was taken immediately after the inoculation of the test organism. Stability of the temperature indicator was used as a guide to complete mixing of the test inoculum in the digesting waste. This usually took about 10 s after inoculation (which itself took approximately 10 s to complete). Samples were rapidly cooled in an ice-bath and, after a 1:10 serial dilution in sterile tryptone soy broth, samples were plated by the standard spread plate method using 0·1 ml of dilution on appropriate media. Campylobacter jejuni samples were diluted in brucella broth. In addition to the standard spread plate enumeration method, pour plates were established by using 1 ml of the direct sample 15–30 min after the start of the experiments, to increase efficiency of recovery of the test inoculum.

The media and growth conditions employed for the recovery of test populations from pasteurization studies are shown in Table 1. Plates were set up in triplicate and all those with growth counted. Media were obtained from Oxoid.

Table 1.  Media and growth conditions employed to recover test organisms from pasteurization studies using a model agricultural waste in a batch thermophilic aerobic digestion process carried out in a 1·5 l stirred tank reactor
Test organismMedia used (Oxoid)Incubation temperature (°C)/time/conditions
Escherichia coliMcConkey agar, eosine methylene blue agar, plate count agar37/18–36 h
Enterococcus faecalisMcConkey agar no. 2 (incubation in candle jar)37/18–36 h
Salmonella enteritidisMcConkey agar, plate count agar37/18–36 h
Campylobacter jejuni Campylobacter blood free agar + CCDA selective supplement (SR 155); incubated42/48–72 h in enriched CO2 atmosphere generated with Campylobacter gas-generating system
Pseudomonas aeruginosaPseudomonas agar base + supplement (SR 103 or SR 102), plate count agar37/18–36 h
Serratia marcescensMcConkey agar, plate count agar30/18–36 h

Effect of waste suspended solids content on pasteurization

Self-heating of wastes in TAD depends on the amount of total solids in the waste, which in turn affects the amount of biodegradable solids and the amount of ballast water. Suspended solids affect waste pasteurization because of their ability to protect pathogens from thermal inactivation (Shuval et al. 1991). To study the effects of solid content on inactivation of microbial pathogens in TAD, model wastes were formulated with different amounts of suspended and degradable waste solids. Dried corn cob was pulverized in a Waring blender (Waring, MA, USA) and sieved through a 0·25 mm sieve. It was then added to the starch mineral medium at levels of 0, 2, 4 and 6% (w/v) solids, to give a total of 2 (starch only), 4, 6 and 8% (w/v) total solids. This represented the range of solids that may be expected to be encountered in a typical TAD process. Prior to use in the medium, the blended corn cob was treated with 1 n NaOH using a method described by Pece et al. (1994) to promote homogeneous suspension in an aqueous medium. In addition, this treatment also encourages bacterial degradation of the hemicellulose content of the waste by the partial solubilization and removal of lignin, and simulates treatment processes as regularly applied to wastes of cellulosic nature. Studies on the effect of solids on pasteurization were carried out at 55 and 60 °C and pH 7, using E. coli as the representative indicator.

Analysis of results

The results of the inactivation studies are presented as a plot of log10 viable count against time. Decimal reduction times (D-values) of the test population were calculated from a regression of log10 survivors vs time over at least three log cycles during the phase of rapid inactivation. Data used for calculation of the D-values are means of triplicate counts and D-values are the means of at least two independent experiments. Means of D-values were subjected to anova using the InstatR (Statistical Services Centre, University of Reading, UK) statistical package.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bioreactor and reaction conditions
  6. Composition of starch mineral medium
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Effect of process temperature and pH on inactivation of populations of test bacteria

The decimal reduction data (D-values) for test isolates are given in Table 2. Starting from an initial test population of approximately 107 ml−1, there was a very rapid reduction in the number of viable cells immediately following their inoculation into the digesting waste at all temperatures and pHs. At 60 °C, in 2% total solids (starch simulated waste), E. coli was undetectable after 15 min at pH 8·0 and 20 min at pH 7·0 (Fig. 1a). These represent higher than 6 log reductions in the population of indicator bacteria under the reaction conditions (Fig. 1a). The D-value calculated at pH 7·0 (2·22 min) was significantly (P < 0·05) higher than that at pH 8·0 (1·42 min, Table 2). There was no regrowth of populations and viable cells were not detected at any point up to 24 h.

Table 2.  Effect of digestion temperature and pH on the D-values of test populations of bacteria during agricultural waste in a batch thermopholic aerobic digestion process carried out in a 1·5 l stirred tank reactor
 Sterile control treatmentsTest treatments (digesting model waste)
 Buffer pH 7·0Starch pH 7·055 °C60 °C
Test organism55 °C60 °C60 °CpH 7·0pH 8·0pH 7·0pH 8·0
  • *

    Standard deviation of the mean of three replicate determinations.

  • Control treatments were set up with Escherichia coli in sterile phosphate buffer at pH 7·0 and in sterile model waste also at pH 7·0.

E. coli3·94 ± 0·27*2·10 ± 0·062·10 ± 0·104·52 ± 0·424·83 ± 0·212·22 ± 0·041·42 ± 0·09
R20·8920·9740·9880·8910·8070·8810·933
Enterococcus faecalis   8·30 ± 0·046·61 ± 0·744·72 ± 0·575·24 ± 0·46
R2   0·6620·9270·9290·95
Salmonella enteritidis   5·03 ± 0·38 0·46 ± 0·35 
R2   0·810 0·852 
Campylobacter jejuni   0·99 ± 0·04 0·71 ± 0·02 
R2   0·979 0·960 
Pseudomonas aeruginosa   5·53 ± 0·323·17 ± 0·072·61 ± 0·183·00 ± 0·17
R2   0·7930·8750·7850·841
Serratia marcescens   6·23 ± 0·967·80 ± 0·953·46 ± 0·395·12 ± 0·25
R2   0·8750·8970·8230·95
image

Figure 1. Effect of temperature and pH on the inactivation profile of some indicator bacteria. (a) Escherichia coli and (b) Pseudomonas aeruginosa in a model agricultural waste during batch aerobic thermophilic digestion process carried out in a 1·5 l stirred tank reactor. ○, 55 °C, pH 7; ●, 55 °C, pH 8; ▵, 60 °C, pH 7; ▴, 60 °C, pH 8

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At 65 °C inactivation was very rapid with similar D-values at both pH 7 and 8 (0·32 min), leading to complete inactivation of the population in 10 min. A reduction in viable populations of more than 5 log values was obtained during inactivation at 55 °C in the first 25 min following inoculation. A slightly higher D-value (4·52 min) was obtained at pH 7 than at pH 8 (4·83 min), but there was no significant difference in the D-values at this temperature. Following the rapid initial decline, inactivation at 55 °C followed a more gradual course providing a stable shoulder, with viable populations persisting up to 60 min (Fig. 1a). However, no viable populations were recovered after 90 min. A slightly lower population persisted at pH 7 than at pH 8 at both 55 and 60 °C. There was no difference in E. coli inactivation rate and D-values between waste digested at pH 7·0 and the control sterile phosphate buffer, at pH 7·0, but a more rapid inactivation was obtained in pH 7·0 buffer than in waste digesting at pH 8·0 at both temperatures.

The inactivation of Ps. aeruginosa in starch mineral medium followed a profile similar to that of E. coli for an initial 10 min at 55 and 60 °C. A 4 log reduction in population was achieved in the first 20 min at both temperatures (Fig. 1b). Decimal reduction times during the linear (first order death) inactivation period of the studies are shown in Table 2. Following an initial rapid inactivation, the rate reduced leaving a resistant shoulder. At 55 °C, up to 10 cfu ml−1 of waste could be recovered after 24 h. Significantly greater (P < 0·05) inactivation was obtained at pH 8·0 than at pH 7·0 during digestion at 55 but not at 60 °C, during which similar inactivation was obtained (P > 0·05). Following the initial rapid decline in populations at 60 °C, viability levelled off at about 100 cfu ml−1 after the first 2 h. Viable populations were recovered from waste digesting at 60 °C for up to 6 h but not after 12 h, or longer, whereas complete inactivation was obtained within 30 min at 65 °C.

Inactivation of Ent. faecalis was less rapid than was the case for either E. coli or Ps. aeruginosa. Rapid inactivation took place within the first 20 min at both 55 and 60 °C with viable populations dropping to about 104, approximately representing a 3 log reduction in the viable population (Fig. 2a). Thereafter, viable cells of up to 10 cfu ml−1 remained in the digesting waste for up to a further 60 min at 60 °C but not longer (Fig. 2a). Viable cells could not be recovered from the digesting waste after 2 h at 60 °C. Inactivation was significantly (P < 0·05) quicker at pH 8 than at pH 7·0 during inactivation at 55 °C. However, there was no significant difference in the inactivation rate during digestion at pH 7·0 or 8·0 and 60 °C. Viable populations of 102 cfu ml−1 remained after 12 h and up to 10 cfu ml−1 could be recovered after 24 h at 55 °C. Table 2 shows the decimal reduction times of the populations at the temperature and pH tested.

image

Figure 2. Effect of temperature and pH on the inactivation profile of some indicator bacteria. (a) Enterococcus faecalis and (b) Serratia marcesens in a model agricultural waste in a batch aerobic thermophilic digestion carried out in a 1·5 l stirred tank reactor. ○, 55 °C, pH 7; ●, 55 °C, pH 8; ▵, 60 °C, pH 7; ▴, 60 °C, pH 8

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Inactivation studies with Ser. marcescens indicated that this organism was considerably more resistant to thermal inactivation than the other members of the Enterobacteriaceae and comparable to Ent. faecalis (Fig. 2b). The inactivation at 55 °C and pH 7·0 followed a profile similar to that of other organisms, with viable cells decreasing rapidly in the first 20 min of exposure (Fig. 2b). However, up to 102 cfu could be recovered after 24 h. At 60 °C and pH 7, up to 10 cfu remained in the digest after 12 h. Decimal reduction values for this organism are also shown in Table 2. There was no significant difference in the decimal reduction values obtained at pH 7·0 and 8·0 at 55 °C but D-values were significantly higher (P < 0·05) at pH 8·0 and 60 °C than at pH 7·0.

Salmonella enteritidis exhibited considerable thermal resistance at the temperatures in this study (Table 2). There was an initial 3 log decrease in the population of viable cells within the first 10 min of heat treatment, followed by a period of stability in the population of viable cells, lasting for up to 60 min at 55 °C before decreasing gradually for a further 12 h to less than 10 cfu ml−1 of digesting waste. No viable populations could be recovered after 24 h at 55 °C. Complete inactivation of viable cells was obtained at 60 °C in 2 h following an initial 4 log reduction in the viable population in the first 30 min.

Campylobacter jejuni was the most heat-sensitive of all the organisms used in this study. Rapid inactivation was obtained at 55 and 60 °C at pH 7·0 (Table 2).

Effect of suspended solids content on the inactivation of Escherichia coli at 55 and 60 °C

Increasing the concentration of suspended solids in the digesting waste led to a considerable increase in the recovery of the test organism at both 55 and 60 °C (Fig. 3a,b). At 4% (w/v) solids level and 55 °C, viable populations of up to 102 cfu ml−1 could be recovered at 18 h and more than 10 cfu μl−1 remained after 24 h compared with the complete inactivation obtained after 90 min in the 2% (w/v) starch medium. At 8% (w/v) solids, up to 102 cfu remained after 24 h. A similar profile of solids protection of the population was obtained at 60 °C with only slightly increased inactivation at 6 and 8% (w/v) solids as compared with 55 °C (Fig. 3a,b). Complete inactivation of E. coli at 4% (w/v) solids was only obtained after more than 12 h at 60 °C (data not shown).

image

Figure 3. Effect of waste suspended solids contents on the inactivation profile of Escherichia coli at different temperatures. (a) 55 °C and (b) 60 °C in a model agricultural waste. Per cent solids (pH 7): ○, 2%; ●, 4%; ▵, 6%; ▴, 8%

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bioreactor and reaction conditions
  6. Composition of starch mineral medium
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

Effective reduction in the pathogen load of waste sludge treated by TAD has been demonstrated in pilot-scale operations (Kabrick & Jewell 1982; Carrington et al. 1991; Kelly et al. 1993). However, these pasteurization data have been based on the background level of pathogens and indicators present in the wastes, which is highly variable depending on the nature and source of the waste. Similarly, the presence/absence test has been used principally in following the pasteurization. This is the first time, to our knowledge, that a systematic evaluation of the inactivation capacity of a TAD process using D-values, an approach which has previously been limited to the food industry, has been applied in monitoring waste pasteurization. It is expected that this should make comparison of inactivation data between treatments less difficult in future.

Carrington et al. (1991), Simpson et al. (1994), Kelly et al. (1993) and Kabrick & Jewell (1982) have reported anomalous and thermoduric tendencies in the inactivation behaviour of Streptococcus spp. in both waste and dairy products, with numbers of indicators in the treated waste exceeding those in the feed waste even though thermophilic temperatures (68 °C) were achieved. Although the authors did not speculate on the possibility of growth of the populations at the treatment temperature, it is important to compare their observations with the considerable heat resistance of the organisms employed in this study. Craven et al. (1997) suggested that faecal streptococci should not be employed as indicators in food because of anomalous heat resistance behaviour. Although re-growth of the indicator mesophilic population is not likely to be a major problem in well mixed systems such as TAD, the behaviour of Enterococcus in this and other published works emphasizes the need for caution in the interpretation of indicator inactivation data and the need to choose indicator populations carefully. Shuval et al. (1991) noted that faecal streptococci could survive for extended periods in thermophilic composting, long after members of the Enterobacteriaceae, including Salmonella, have disappeared.

Of interest in this study is the tendency of the initial rapid inactivation of populations to cease after a few minutes, particularly at lower temperatures. This led to extended persistence of viable populations as indicated by the tail or shoulder (Fig. 1). It is possible that agglomeration of cells and attachment to particulate materials in the waste led to an extended heat resistance. Botner (1991) reported an extensive and highly resistant tail during the test inactivation of Aujeszky's disease virus in animal waste slurry. The tail was thought to be due to a small, highly resistant, core population. Whether a similar phenomenon exists in bacteria is not known. Protection of pathogens including Salmonella and E. coli by milieu particles has been reported by Strauch et al. (1985) and Kornacki & Marth (1993a,b) and in Ascaris suum eggs by Plachy et al. (1995). This protective effect becomes less significant as the heating temperature increases. A proportional increase in the protective effect of solids in waste treatment on enteric viruses, irrespective of the treatment temperature, has been reported by Ward & Ashley (1978). In this work, E. coli was considerably protected by the increase in the waste solids, with protection reducing as the test temperature increased.

Conflicting reports exist on the effect of pH on the heat sensitivity of pathogens and indicators. Plachy et al. (1995) reported enhanced heat sensitivity of parasite eggs with an increase in pH. However, this effect was reduced as the digestion temperature dropped towards a mesophilic range (Juris et al. 1992; Plachy et al. 1993). The effect of pH on the inactivation of test populations in this study is slightly conflicting and anomalous. Whereas E. coli was more sensitive to heat at pH 8 and 60 °C, there was no difference due to pH at either 55 or 65 °C. However, for Enterococcus there was no difference in death rate due to pH at 60 °C, but sensitivity was greater at pH 7 at 55 °C. Pseudomonas and Serratia behaved similarly in being more sensitive to heat at 60 °C at pH 7 than pH 8. At 55 °C the effect of pH was reversed for Pseudomonas, behaving similarly to Ent. faecalis, while there was no difference in sensitivity at this temperature in Serratia.

Simpson et al. (1994) reported that slightly alkaline conditions increased the D-values of Streptococcus faecium. A similar observation was made concerning the heat sensitivity of Listeria monocytogenes in eggs whereas Salmonella was reportedly more heat-sensitive at pH 9·3 than at 7·8 (Palumbo et al. 1995, 1996). It appears that the effect of pH on heat sensitivity depends on the heating menstruum and the test population.

The role of oxygen tension in the pasteurization of waste is not well understood. However, the period of low DO content in the digesting waste would normally be associated with periods of high volatile acid content in the waste, and this has variously been reported to enhance the heat sensitivity of pathogens. Kearney et al. (1993a,b) reported that high VFA content in animal slurry undergoing anaerobic digestion helped to improve the inactivation of Salmonella. It has also been reported that VFA such as acetate improve the efficacy of waste pasteurization (Phae et al. 1996; Mcintosh & Oleszkiewicz 1997). However, A. suum eggs were equally deactivated at thermophilic temperatures (55 °C) in both aerobic and anaerobic processes (Juris et al. 1992).

The application of TAD for pasteurizing waste sludge is on the increase and, with increased intensive farming, this trend will go on and efforts at improving pasteurization of slurry are likely to continue. Hence, studies designed to understand the inactivation efficiency of TAD are of considerable importance. Droffner & Brinton (1995) observed the survival of E. coli and Salmonella for up to 9 d at 60 °C, making the requirement for inactivation data in waste treatment processes more urgent.

The conditions selected for this study cover the range of pH, temperature and solid contents that may be expected to be encountered in full-scale TAD processes in the hope that the data may be employed in optimizing waste pasteurization in wet composting.

The application of D-values for assessing microbial inactivation makes the comparison of inactivation data resulting from different treatment processes straightforward. The extensive protection of test populations due to the increase in waste solids implies that pasteurization of high solid slurries by thermophilic digestion will require increasingly longer exposure of the waste to high temperature. The extended resistance of Enterococcus to heat suggests that considerable thought be given to the selection of this organism as an indicator in treatment processes where the inactivation of more sensitive organisms is the process target.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bioreactor and reaction conditions
  6. Composition of starch mineral medium
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References

The authors wish to thank the British Commonwealth Commission for their support during this study and Kellogg Co. Ltd. (Seaforth Coalmills) for kindly supplying the corn cobs.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bioreactor and reaction conditions
  6. Composition of starch mineral medium
  7. Results
  8. Discussion
  9. Acknowledgements
  10. References
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