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Keywords:

  • composting;
  • hygienization;
  • micro-organisms;
  • pathogens;
  • sludge

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To study the decrease of enteric micro-organisms including viable nematode eggs, enteroviruses, faecal indicators (Escherichia coli and enterococci) and pathogenic bacteria (Listeria monocytogenes, Salmonella sp. and Clostridium perfringens) of a rural sewage sludge when it is composted for 7 months in mixture with straw.

Methods and Results:  Numbers of the test organisms and the physico-chemical parameters were measured on a monthly basis on the mixture, on the compost after being turned, and on the pile in three positions representing the part by which air is incoming, the bottom of the pile and the part through which air is outgoing. The lowest temperature in the pile was observed at the bottom, where it did not exceed 50°C against 66°C in the two other areas. There were no significant differences between the three areas in terms of micro-organism survival. Infectious enteroviruses were inactivated rapidly and were not found after the first turning whereas some genomes were detected until after the third turning. Escherichia coli and enterococci presented a similar survival rate and their number decreased by 4 log10 whereas Salmonella decayed at a greater rate than L. monocytogenes. The numbers of C. perfringens decreased gradually to reach a final concentration in the mature compost of about 102 CFU g−1 dry matter (d.m.), which was similar to that of the faecal indicators.

Conclusions:  The hygienic effect of sludge composting in mixture with straw results in a significant reduction of enteric micro-organisms, the concentration of the faecal indicators in the final product being <64 most probable number g−1 d.m. The concentrations of Salmonella, enteroviruses and viable nematode eggs in the final product were not detectable which is in accordance with the French legislation.

Significance and Impact of the Study:  The results which pointed out the different behaviour of the test micro-organisms reflect the difficulty to propose a relevant indicator of hygienization. Otherwise, they show that composting is an efficient means for hygienization of sludge of rural wastewater treatment, where the straw is available close to their place of production.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Land application and incineration are the main routes for sewage sludge from wastewater treatment plants in France. As incineration is not economically viable for small wastewater treatment facilities, land application is the main means used for recycling their sludge at present. To minimize the potential microbiological risk for the health of humans, animals and plants, sludge should not be applied continuously and must be stored in tanks for about 6 months. Since 1998 (decree of 8 January), the French environmental regulations have defined ‘sanitized sludge’ as sludge that has been treated so that pathogens are no longer detectable (Anon 1998). Then, for sludge to be recycled to agricultural land, it must comply with the following numerical limits: for Salmonella sp., the Most Probable Number (MPN) of cells must be less than 8 per 10 grams total solids (dry weight); for viable nematode eggs, the MPN of eggs, <3; and, for enteric viruses, the most probable cytopathic number, <3. In another way, the decree recommends the enumeration of faecal coliforms. To provide a hygienically safe product to the farmers, it became therefore necessary to explore alternative or complementary methods to storage which is the main treatment of sludge from rural wastewater treatment plants. Composting appears to be a promising way to make such a product from the sludge of small facilities because of its low cost and no advanced technology is required.

As the most prevalent group of pathogenic micro-organisms in sludge is enteric pathogens (Hay 1996), most of the studies evaluating hygienization of composting of sludge or waste were restricted (i) to the faecal indicators, i.e. faecal streptococci, faecal coliforms or Escherichia coli (Soares et al. 1995; Vuorinen and Saharinen 1997; Sesay et al. 1998; Tiquia and Tam 2000; Turner 2002), (ii) to Salmonella, which was used as a representative pathogen for modelling die off of enteric pathogenic micro-organisms (Hu et al. 1997; Tiquia et al. 1998; Sidhu et al. 2001), or (iii) to both faecal indicators and Salmonella (Pereira-Neto et al. 1986; Droffner and Brinton 1995). Only a few more general studies have reported, besides the behaviour of faecal indicators and Salmonella, the survival of other types of micro-organisms such as Yersinia enterocolitica, toxigenic E. coli, Shigella, staphylococci, sulfite-reducing anaerobe spores or nematode eggs (Goldstein et al. 1989; Deportes et al. 1998; Gantzer et al. 2001; Hassen et al. 2001). Furthermore, Droffner and Brinton (1996) and Böhnel and Lube (2000) have respectively studied the behaviour of Listeria monocytogenes and Clostridium botulinum during composting.

The present study examines the hygienization of sludge from a rural water treatment plant for 2000 equivalent-inhabitants during the composting process. It deals with the composting of a mixture of wheat straw (in a quantity as small as possible) and pressed sludge, a mixture adapted to small districts which have both these substrates at their disposal. It covered one whole compost cycle lasting from freshly pressed sludge to mature compost over a period of 7 months. Chemical and physical parameters which could have an influence on the survival of pathogens were analysed in the initial mixture and in the compost after turning and before turning at three relevant positions on the pile, including the side of the incoming air flow (area 1), the bottom in the middle of the pile (area 2), and the top of the pile (area 3), area through which air is outgoing. The inactivation of three types of micro-organisms was monitored: (i) bacteria (faecal indicators, Clostridium perfringens, Salmonella sp., Listeria sp. and L. monocytogenes), (ii) viable eggs of nematodes, and (iii) enteroviruses.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Raw material

Sludge came from a storage tank of the rural wastewater treatment plant of Ecouflant (a village near Angers, Maine-et-Loire, France) which employs a conventional activated sludge system. The sludge which initially contained 3% of dry matter was pressed prior to composting using a filter band to obtain 15% of dry matter. Wheat straw came from a farm at Corseul, Côtes d'Armor, France. The chemical and microbiological characteristics of the sludge and the straw used for the experiment are presented in Tables 1 and 2.

Table 1.  Chemical characteristics of sludge and straw used for composting
MatrixpHMoisture (%)Total N (mg g−1 d.m.)C/NOrganic C (mg g−1 d.m.)
  1. Values are mean (SD).

Straw8·2 (0·2)15·1 (0·9)6·8 (0·7)54·3 (4·7)365·0 (7·2)
Sludge6·8 (0·0)97·1 (0·1)60·6 (1·4)4·4 (0·2)269·7 (17·1)
Table 2.  Ranges obtained for four samples or detection limit of concentrations of micro-organisms in sludge and in straw used for composting
MatrixBacteria (cells or MPN g−1 d.m.)Entroviruses (g−1 d.m.)
E. coliEnterococciC. perfringensListeria sp.L. monocytogenesSalmonella sp.Genome copiesMPNCU
  1. *The symbol ‘<’ indicates the detection limit of the method. MPNCU, most probable number of colony unit.

Straw<5*<5<23<0·2<0·2<0·2<67<0·7
Sludge4·4 × 105–1·1 × 1067·2 × 105–2·6 × 1064·5 × 106–1·9 × 1075·2–3·8 × 1023·8–3·8 × 1021·2–3·24·5 × 103–2·5 × 10415–80

Construction of the heap

The composting was carried out at Coëtfinet, Corseul, Côtes d'Armor, France, in the ‘Centre d'Aide par le Travail de 4 Vaulx’. It involves an open greenhouse with a concrete floor on which boxes are installed 6 m long and 4 m wide, which are able to be filled up to 2 m in height, separated by concrete walls heightened by planks. A total of 8·1 tonnes of pressed sludge was mixed with 1·4 tonnes of straw in a 1 : 0·17 ratio based on the weight. The C : N ratio of the starting compost material was 9·3 : 1, the moisture content, 74·9%. No moisture adjustment was carried out thereafter. A homogeneous mixture of the components was made possible by the use of a mixer weigher. The mixture deposited on the ground was then put into a spreader in order to expand it (thus ensuring aeration) and to set it up in the box. The mixture was composted in a trapezoidal-shaped pile (1·2 m high with bases of 4·85 and 3·85 m and 3·90 m wide). A cover was placed about 0·5 m above the pile, with two ventilation pipes set in it in order to allow air circulation (Fig. 1). This setup, completed with an exterior fan, aimed at permitting the measurement and quantification of the gaseous emissions, thanks to establishing an air circulation direction. The air flow was fitted to mimic natural conditions, i.e. when the ambient air drags the moisture and the calories without hindrance. The composting period carried out for the first 4 months, with turning every month, was considered as the fermentation phase, and the following 3-month period without turning, as the maturation phase. The cover and shafts above the heap were removed before turning and put back after turning. During turning, the pile was mixed thoroughly to ensure sufficient aeration before reformation.

image

Figure 1. Schematic description of the pile and sampling positions: 1, part of the pile by which air is incoming; 2, bottom; 3, part through which air is outgoing. Arrows indicate the air flow

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Sampling

The straw was mixed with the sludge on 26 March. Samples were taken on a monthly basis: t0 (26 March), t1 (23 April), t2 (14 May), t3 (11 June), t4 (17 July) and at the end of maturation (t5: 15 October). Sampling (about 15 kg being collected) was carried out in three positions of the pile (Fig. 1), representing the part by which air is incoming (area 1 located about 0·4 m from the side of the incoming air flow), the bottom (area 2 located about 0·2 m from the base of the pile), and the part through which air is outgoing (area 3 located about 0·4 m from the outer surface of the pile) and on the mixture being turned. Collected matter was mixed and repetitively divided in half, until homogeneous samples of approx. 1 kg were obtained. The divisions were carried out using a sampler divider, composed of a rectangular metal chute with a divider in the middle. The samples were stored in closed bags, and either kept for physico-chemical parameters analyse, or placed at 4°C before being analysed for one of the studied parameters. The samples were stored <24 h prior to microbiological examination. Sampling was performed using the same methodology for the substrates, except that the sample divider could not be used for straw. Four samples of straw and sludge were analysed prior to composting. Then, four samples were taken during composting in each position of the pile and from a mixture of compost.

Physico-chemical parameters

The pile temperature profile was recorded by means of thermocouple probes inserted in the three areas of the pile and connected to a digital logger. The temperature was measured every hour. Moisture was determined by drying at 100°C for 48 h. The pH was measured in the aqueous extract of the compost sample with distilled water (1 : 5, w/v). Total Kjeldahl nitrogen and total organic carbon were estimated by using the ISO recommendations (Anon 1999a,b).

Microbial analysis

Detection and quantification of viable nematode eggs.  Viable eggs of helminths were numerated according to the US Environmental Protection Agency recommendation (Anon 1992). Samples were processed through a series of steps (sedimentation, flotation on a density gradient, passage through sieves, and a phase separation). Viability of eggs was estimated after incubation at about 28°C for 1 month. After incubation, eggs that contain a larva were counted as viable.

Detection and quantification of enteroviruses.

Virus extraction.  Viruses were eluted from the different matrices (quantities equivalent to 5 g of dry matter) according to the technique of Ahmed and Sorensen (1995). A quantity of 45 ml of 10% beef extract (pH 9; LP029B; Oxoid, Dardilly, France) was added and the mixture was stirred at 700 oscillations min−1 with the Flash Shaker SF-1 (Sigma Aldrich, St Quentin, France), sonicated on ice (100 W) for 5 min with 1-min bursts, mixed for 5 min, and then centrifuged at 5000 g for 1 h at 4°C. The supernatant, adjusted to pH 7·2, constituted the extract.

Virus concentration.  For virus concentration, we used polyethylene glycol 6000 precipitation as described by Lewis and Metcalf (1988); 8% (w/v) polyethylene glycol 6000 (in a phosphate solution at pH 7·2) was added. After agitation at 700 oscillations min−1 with the Flash Shaker for 5 min, the mixture was kept at 4°C overnight and then centrifuged at 10 000 g for 90 min at 4°C. The pellet, suspended in 12 ml of phosphate buffer (pH 7·2), constituted the concentrate; as a final step, the pellet was decontaminated by adding 4 ml of chloroform. This concentrate was used for cell culture and viral RNA extraction.

Cell culture.  Infectious enteroviruses were counted by inoculating the decontaminated concentrates into buffalo green monkey (BGM) cell cultures in 96-well microplates, as described by Monpoeho et al. (2001). The results were expressed using the Most Probable Number of Colony Unit (MPNCU) per gram (dry weight) of matter.

Viral RNA extraction.  Enterovirus RNA was extracted from 250 μl of concentrate with an Rneasy plant mini kit (Quiagen, Courtaboeuf, France) according to the manufacturer's instructions. However, a modified lysis buffer containing 2% (w/v) polyvinylpyrrolidone PVP 40 000 (Sigma) was used.

Fluorogenic RT-PCR.  To design the primers and probe used for the TaqMan technique, the most constant genome region in enteroviruses, the 5′ noncoding region was chosen (Monpoeho et al. 2000). The real-time fluorescence was measured as described by Monpoeho et al. (2001).

Quantification of C. perfringens. Clostridium perfringens were counted using the AFNOR method (Anon 1999c). Ten grams of the sample were added to 90 ml of a sterile tryptone salt medium (Biokar, Beauvais, France). One millilitre of a serial dilution (in tryptone salt medium) was then transferred into a Petri dish and covered by tryptose sulfite cycloserine medium (Biokar) maintained at 47°C. After incubation at 37°C for 20 h under anaerobic conditions, typical black colonies were transferred into thioglycholate broth (Biokar) and incubated at 37°C for 20 h under anaerobic condition. Five drops of the seeded thioglycholate broth were then transferred into a tube of lactose sulfite broth (Biokar) and incubated at 46°C for 24 h. Bacteria developing in the lactose sulfite broth with H2S production leading to a black precipitate were considered as C. perfringens. Bacterial counts were determined by the numbers of black colonies on tryptose sulfite cycloserine which produce H2S on lactose sulfite broth. Concentrations were expressed as CFU g−1 dry matter (d.m.).

Quantification of enterococci and E. coli.  Enterococci and E. coli were respectively enumerated by using MUD/SF (Anon 1999d) and MUG/EC (Anon 1999e) microplates techniques. Two grams of the sample were added to 18 ml of a sterile special microtitration plate diluant (Bio-Rad, Marnes-la-Coquette, France). After hand shaking for 1 min, the samples were then serially diluted (10−1, 10−2 and 10−3) in the diluant and 200 μl of each dilution were inoculated into 24 wells of the microplates MUD/SF and MUG/EC (Bio-Rad). After an incubation of 36 h at 44°C, the microplates were observed under UV light (360 nm) to detect specific enzymatic reactions from enterococci and E. coli. Concentrations of faecal indicators were expressed as MPN g−1 d.m.

Quantification of L. monocytogenes.  Enumeration was performed with the MPN technique using three tubes per dilution. Amounts of 10 g of the matrix were transferred into 100 ml of the modified Fraser broth previously described by Garrec et al. (2003a), and amounts of 1 g or 0·1 g were transferred into 10 ml of the modified Fraser broth. After incubation at 37°C for 48 h, 0·1 ml of an appropriate dilution of each enrichment broth was plated onto Palcam agar (Biokar). After an incubation of 48 h at 37°C, presumptive Listeria colonies of i.e., grey-green colonies with a black sunken centre and a black halo, were transferred onto Rapid'L.mono agar plates (Bio-Rad) using a 90 mm diameter nitrocellulose membrane (Millipore, Saint Quentin en Yvelines, France). The number of colonies transferred ranged between 1 and c. 200. After an incubation of 24 h at 37°C, characteristic colonies of L. monocytogenes i.e. blue colonies, were confirmed by their phosphatidyl inositol phospho-lipase C activity. Concentrations of L. monocytogenes were expressed as MPN g−1 d.m.

Quantification of Salmonella.  Enumeration was performed with the MPN technique using three tubes per dilution. Amounts of 10 g of the matrix were transferred into 100 ml of the pre-enrichment selenite-cysteine broth (Biokar) and amounts of 1 g or 0·1 g were transferred into 10 ml of the pre-enrichment broth. After incubation at 37°C for 24 h, a volume of 0·1 ml of each pre-enrichment broth was then transferred to 10 ml of Rappaport–Vassiliadis broth (Biokar) and incubated at 42°C. After 24 h of incubation, one loopful (10 μl) from each broth was streaked onto Rambach agar plates (Bio-Rad). The plates were incubated at 37°C for 24 h and examined for the presence of presumptive Salmonella colonies, i.e. fuchsia-coloured colonies. Presumptive colonies of Salmonella were isolated on nutrient agar and identified with API 20E strips (bioMérieux, Marcy l'Etoile, France). Concentrations of Salmonella were expressed as MPN g−1 d.m.

Statistical analysis

To compare variations in composting during the process and at different positions of the pile and in the mixture of compost, a principal components analysis was performed using STABOX pro 5 (added to Excel). For this analysis, logarithmic values of the bacterial and virus concentrations were used as microbial parameters, and subsequently, mean values of the physical, microbial and chemical parameters at the three positions sampled in the pile and in the mixture of compost were compared using the Newman–Keuls test. A P-value below 0·05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Physical and chemical data

Temperature profiles during the fermentation and maturation phases of composting measured in three positions of the pile are shown in Fig. 2. During the fermentation phase, temperatures of both areas 1 and 3 were higher and presented a different evolution from those observed at the bottom of the pile. The temperature maxima of these areas were rapidly reached after each turning (in 2–4 days) and ranged between 53·6 and 69·4°C, whereas the temperature at the bottom reached 49·8°C only in the second month and did not exceed 41·7°C otherwise. Between each turning and after these maxima had been reached, the temperature dropped slowly and continuously. It remained however relatively high, especially in area 1 where it exceeded 55°C for 5–16 days according to the period of the active composting. During the maturation phase, the temperature still reached 55°C in area 3. On the whole, the high temperature maintenance in the three positions of the pile revealed a significant respiratory activity of the micro-organisms.

image

Figure 2. Temperatures inside the heap at 0·4 m depth (area 1; area 3), and 1 m depth (area 2, bottom of the pile). Days 0, 27 (1st month), 48 (2nd month), 76 (3rd month), 111(4th month), and 202 (end of maturation) correspond respectively to the settling, turnings and unloading of the heap. Arrows indicate turning event. bsl00088, Area 1 (incoming air flow); inline image, area 2 (bottom); inline image, area 3 (outgoing air flow)

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During the fermentation phase, the moisture content measured at each turning decreased from 74·9 to 64·4%. It reached 60·5% after the maturation phase (Table 3). Greatest moisture was always observed in area 2 because water at the bottom was less extracted by the circulating air. Due to a too weak circulation of air in the pile, not enough water was eliminated during the second month of composting. However, the moisture which evolved between 74·5 and 65·5% corresponded to a range providing a maximum activity of micro-organisms present in the compost, according to Liang et al. (2003). This draining lack was corrected afterwards by increasing the air flow leaching the pile.

Table 3.  Chemical and physical characterization of the three tested areas of the pile during composting and in the mixture (after turning)
TimeZonepHMoisture (%)Total N mg g−1 d.m.C/NOrganic carbon mg g−1 d.m.No. days >45°CNo. days >55°CTemp. max*
  1. Values are mean (SD). n.d., not determined.

  2. *Maximal temperature observed in the position.

t0Mixture6·7 (0·0)74·9 (0·6)32·7 (0·6)9·3 (0·2)305·7 (2·9)   
1st month (t1)18·1 (0·2)70·2 (2·5)38·9 (7·8)7·7 (1·4)290·7 (1·3)24·116·563·2
28·1 (0·2)76·0 (1·1)33·2 (2·8)10·4 (0·4)344·1 (40·1) 0 041·7
37·7 (0·3)67·6 (7·2)39·8 (4·0)7·6 (1·6)297·7 (42·1)21·9 5·066·6
Mixture7·8 (0·2)70·8 (1·1)37·9 (5·8)9·3 (1·8)333·1 (20·9)   
2nd month (t2)16·9 (0·7)65·5 (1·9)33·4 (6·0)8·0 (1·4)261·1 (7·4)20·515·269·4
28·4 (0·1)74·5 (1·1)40·3 (3·8)7·3 (0·6)292·1 (15·0)17·5 049·8
38·3 (0·2)71·5 (5·5)32·9 (3·1)8·9 (0·8)291·7 (5·3)20·219·567·0
Mixture8·3 (0·2)72·7 (1·3)37·9 (4·1)7·5 (0·8)282·1 (6·6)   
3rd month (t3)16·6 (0·6)67·2 (2·3)35·0 (2·2)8·3 (0·5)290·6 (16·8)281663·6
28·6 (0·0)72·5 (0·7)39·9 (1·7)7·1 (0·4)283·6 (8·8) 0 038·9
38·6 (0·1)70·6 (1·5)41·4 (2·2)7·3 (0·5)303·0 (11·5)18 053·3
Mixture8·0 (0·4)70·6 (1·7)39·5 (11·1)7·9 (3·1)284·9 (7·6)   
4th month (t4)16·1 (0·2)65·5 (1·0)32·7 (2·7)7·7 (0·7)252·4 (18·0)29·5 7·856·7
27·7 (0·2)70·9 (0·3)31·7 (1·5)7·6 (0·4)239·5 (5·2) 0 040·0
37·8 (0·7)67·6 (0·4)37·3 (3·6)6·8 (0·7)250·4 (7·9)30·6 9·456·9
Mixture7·4 (1·1)64·4 (1·2)n.d.n.d.254·1 (12·7)   
7th month (t5)Mixture4·8 (0·1)60·5 (4·4)31·2 (5·1)7·4 (1·1)226·9 (6·2)   

The concentration of organic carbon did not significantly differ in the three positions. It regularly decreased during composting from 306 to 227 mg g−1 d.m., indicating a continuous degradation of organic carbon. The total nitrogen content, initially 32·7 mg g−1 d.m., remained sensibly the same during active composting and did not significantly differ in the three positions. The initial C/N ratio of the compost (9·3) decreased to 7·9 after 3 months of composting and to 7·4 after the maturation phase, reflecting a more rapid decrease in organic carbon than in nitrogen (Table 3).

The pH, which was initially 6·7, was around 8 in the three positions of the pile after 1 month of composting (Table 3). Afterwards, it differed following the positions. It was significantly lower (P < 0·05) in area 1, where it regularly decreased from 8·1 to 6·1 reflecting a better degradation of the more degradable forms of organic compounds in this area which was the most aerated (production of organic acids by acidogenesis and of CO2, which dissolved in water). In the two other areas, the pH globally increased during the first 2 months of composting to reach 8·6 and then slightly decreased to a value of 7·7 at the end of the fermentation period. This lack of a rapid decrease in pH is characteristic of a lack of oxygenation which is due to a shortage of lacunary spaces consecutive to a too significant moisture. At the end of the maturation, the pH decreased to a low value of 4·8, very likely because of the increase of the nitrates content (data not shown) consecutive to a blocking of the denitrification due to a shortage of available carbon.

Micro-organism densities during composting

Among the three types of micro-organisms followed in this study, only nematode eggs were not detected in the sludge and were never found, afterwards, during the composting. Microbiological analyses of the sludge revealed the presence of Salmonella and L. monocytogenes and of infectious enteroviruses at lower concentrations than 3·2 and 3·8 × 102 MPN g−1 d.m., and than 80 MPNCU g−1 d.m. respectively (Table 2). The effect of composting on the survival of bacteria and enteroviruses (infectious viruses and RNA genomes) is presented in Fig. 3. The enumerations, each undertaken on four samples taken from the mixture during turning, presented fluctuations of about 1 to 2 logarithmic units regardless of the micro-organism. This variability can be attributed to the heterogeneous structure of the matrix and to the nonhomogeneous distribution of the micro-organisms within the compost. The infectious enteroviruses, present at concentrations ranging from 2 to 16 MPNCU g−1 at the beginning of composting, were not found from the first turning regardless on the position of the samplings. However, genomes were detected until the third turning, after which both viral particles and genomes were no longer detected. Faecal indicators, of which the concentrations slightly increased after the first turning (from 4·4 × 106 to 6 × 106 for E. coli and from 8 × 106 to 1·4 × 107 for enterococci), were not totally inactivated although the temperature reached 66°C in the pile. It was necessary to await the second turning to observe a significant decrease (about 2 logarithmic units). Enterococci and E. coli presented a similar length of survival during composting. Indeed, after an increase of their concentration during the first month, both faecal indicators gradually decayed to reach concentrations lower than or equal to 200 MPN g−1 d.m. in the mature product. In addition, both enterococci and E. coli were found in 100 and 95% respectively of the samples carried out at each turning. C. perfringens decayed by approximately a factor 2 until the 4th turning. Systematically present in samples until the third month of composting, C. perfringens was isolated in only one sample of the fourth turning and in three samples of the mature product. After maturation, C. perfringens levels were however comparable with those observed for E. coli (between 50 and 7·6 × 102 CFU g−1 d.m.). Salmonella was present in the four samples of the initial straw-sludge mixture at densities of about 4 MPN g−1 d.m., but was quickly inactivated. It was isolated in only one sample of the first turning at a concentration of 1·5 MPN g−1 d.m., and was not isolated in any of the subsequent sample. The species L. monocytogenes which accounted for 95% of the Listeria cells isolated in the pressed sludge, was still present in the four samples of compost taken after 1 month of composting and accounted for 0·3–5% of the Listeria cells. Listeria monocytogenes and Listeria sp. were detected in one sample and in three samples of the fourth turning respectively.

image

Figure 3. Mean concentrations of target micro-organisms (faecal indicators, Clostridium perfringens, Listeria sp., Listeria monocytogenes, Salmonella and genomes of enteroviruses) during the composting. Bars indicate minimal and maximal values. bsl00116, Echerichia coli; bsl00159, Enterococci; inline image, Clostridium perfringens; —bsl00067—, Listeria sp.;—○—, Listeria monocytogenes; —×—, Salmonella; inline image, Enteroviruses

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Comparison of micro-organism densities in relation to their position in the composting heap

As Salmonella was generally absent in the different samples (always absent in the 2nd month), it was not possible to compare its behaviour depending on its position in the pile. Concentrations of the other micro-organisms did not significantly differ from one area to another (Table 4). As areas 1 and 2 presented similar physico-chemical characteristics, in particular with regard to the water content and the maximum temperature reached, it was not surprising that concentrations of micro-organisms presented the same order of magnitude. However, the bottom of the pile was characterized by a greatest moisture and a temperature which did not exceed 50°C. Otherwise, the frequency of detection of the target bacteria and their concentrations in the mixture analysed at each turning were systematically larger than those of the three areas (Table 4). This phenomenon may be explained by the existence of zones of the compost (crust for instance) in which the concentrations of micro-organisms would be higher. As a consequence, the integral mixture of the pile during each turning may increase bacterial concentrations.

Table 4.  Ranges of concentrations of micro-organisms obtained for four samples in the three areas of the pile during composting and in the mixture (after turning)
TimeZoneE. coliEnterococciC. perfringensListeria sp.L. monocytogenesSalmonella sp.Enteroviruses
N* NPP g−1 d.m. N NPP g−1 d.m. N CFU g−1 d.m. NNPP g−1 d.m. NNPP g−1 d.m. NNPP g−1 d.m. NGenome copies g−1 d.m.
  1. *Number of samples where micro-organisms were detected.

  2. †Not detected, below the detection limit, i.e. <69 for E. coli and enterococci, <5·6 × 102 for C. perfringens (except in area 2 for the 4th month where it was <3·6 × 103), <0·15 for Listeria sp., L. monocytogenes and Salmonella sp., <50 for genome copies of Enteroviruses.

  3. ‡The corresponding MPNCU ranged between 2 and 16 g−1 d.m.

  4. §Due to a technical problem, the area 1 was not analysed.

t0Mixture42·1 × 105–9·2 × 10622·1 × 105–9·6 × 10644·4 × 104–8·4 × 104444 to >4440·8–4441·7–9·642·3 × 103‡–4·8 × 103
1 month (t1)143·4 × 102–3·7 × 10447·1 × 104–1·9 × 10533·4 × 101–6·8 × 10330·5–3·110·12–0·120n.d.3n.d.–1·0 × 103
244·2 × 102–6·3 × 10342·4 × 104–1·9 × 10545·5 × 103–4·2 × 10430·1–0·20n.d.0n.d.42·4 × 102–1·3 × 103
349·1 × 102–3·5 × 10543·8 × 104–8·8 × 10542·5 × 102–3·2 × 10440·2–7·640·1–0·540·9–9·644·9 × 102–1·0 × 103
Mixture42·2 × 106–1·4 × 10741·4 × 106–4·0 × 10746·6 × 103–2·7 × 10424·0–36·040·1–0·51n.d.3n.d.–1·1 × 103
2nd month (t2)143·6 × 103–1·8 × 10445·6 × 103–2·9 × 10441·2 × 103–1·5 × 1041n.d.–0·210 0 41·2 × 102–1·6 × 103
21n.d.†–3·9 × 10343·9 × 102–4·3 × 10543·9 × 103–2·0 × 1042n.d.–0·360 0n.d.3n.d.–1·9 × 103
324·2 × 103–4·2 × 10346·3 × 103–4·9 × 10447·0 × 102–1·1 × 1041n.d.–1·511n.d.–0·320n.d.3n.d.–1·5 103
Mixture42·1 × 103–2·2 × 10447·0 × 104–7·0 × 10543·7 × 103–2·2 × 1043n.d.–0·81n.d.0n.d.44·0 × 102–3·6 × 103
3rd month (t3)11n.d.–2·8 × 10341·2 × 103–1·6 × 1041n.d.–9120·1–0·231n.d.–0·10n.d.0n.d.
20n.d.43·6 × 102–4·4 × 1031n.d.–3·6 × 1042nd -1·561n.d.–0·270n.d.3n.d.–50
31n.d.–652n.d.–4·1 × 1031n.d.–340n.d.0n.d.0n.d.1n.d.–50
Mixture3n.d.–9·2 × 10341·7 × 103–5·1 × 10441·7 × 102–2·0 × 1043n.d.–0·30n.d.0n.d.2n.d.–50
4th month (t4)§20n.d.43·6 × 102–4·4 × 1031n.d.–3·6 × 10420·3–1·61n.d.–0·270n.d.0n.d.
31n.d.–652n.d.–4·1 × 1030n.d.0n.d.0n.d.0n.d.0n.d.
Mixture465–3·4 × 10343·4 × 102–1·0 × 1031n.d.3n.d.–2·610·150n.d.0n.d.
7th month (t5)Mixture2n.d.–2·0 × 1023n.d.–963n.d.–7·6 × 1020n.d.0n.d.0n.d.0n.d.

Microbial behaviour and physico-chemical parameters

The matrix of correlation obtained in multivariate analysis carried out on the microbiological and physico-chemical parameters reveals a significant correlation (P < 0·05) between the concentrations of E. coli and enterococci (R = 0·95) and between the two faecal indicators and Listeria sp. (R ≥ 0·72), which suggests a similar behaviour of these three groups of bacteria during composting. However, none of the physico-chemical parameters had a significant influence on survival of the target bacteria.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The densities of faecal indicators in sludge produced by the wastewater treatment plant and stored for 6 months, which ranged from 105 to 106 MPN g−1 d.m., are comparable with those reported in fresh sludge (Hu et al. 1995; Lucero-Ramirez and Malina 1998; Tsai et al. 1998; Garrec et al. 2003b). No viable nematode egg was detected in the sludge but the concentrations of these parasites are usually very low and ranged from undetectable level to 102 g−1 d.m. (Cadiergues 2000; Gantzer et al. 2001).

The compost made from the sludge of the rural wastewater treatment plant conforms, from the second month of composting, to a hygienized product, according to French legislation, as regards Salmonella, viable nematode eggs and enteroviruses, the concentrations of which were below the limiting values fixed by the decree of 8 January 1998. The hygienic effect of composting sludge with straw reported here is, as well, satisfactory since, in the end product, none of these micro-organisms, and moreover, no L. monocytogenes were detected. The results can be compared with those found in the literature on the impact of composting of sludge on the survival of micro-organisms, and some hypotheses may be advanced to explain the differences between their kinetics of disappearance.

The infectious enteroviruses enumerated between 15 and 80 MPNCU g−1 d.m. in the sludge, were not detectable from the first turning of the compost. This is in agreement with the virus decrease recently reported by Monpoeho et al. (2004) regarding the clearance of viruses from sludge using four stabilization processes. The effect of heat reported by Ward and Ashley (1978a) and Straub et al. (1993) on enteric micro-organisms is also observed in our case as the temperatures during treatment reached 53–69°C that is enough to eliminate enteroviruses. In addition, composting causes a considerable reduction in the concentration of detergents in sludge, whereas several detergents have been shown to be highly protective against heat for enteroviruses in sludge (Ward and Ashley 1978b). Dimmock (1967) had suggested that high temperatures (about 55°C) inactivated poliovirus through damage to the capsid of the virion. More generally, Ward (1984) has shown that the temperature reached during some treatment processes and dehydration could rupture the viral capsid and release nucleid acid. In our study, the genomes of the viral particles undetectable per viral culture from the first turning of the compost, were successfully quantified by RT-PCR up to the third turning. One explanation to this phenomenon would be that the BGM cells used in the viral culture method were not sensitive to all enterovirus species quantified by RT-PCR. Thus Smith and Gerba (1982) reported that BGM cells permitted the replication of coxsackieviruses, whereas echoviruses cultivated with difficulty on these cells. It is however more probable that given the methods of elution and concentration used before the RT-PCR in addition to the detection of an RNA genome (nucleic acid known as fragile), the enterovirus genomes quantified until the third turning of the compost may be encapsided. Their capsids might be damaged or the infectivity of the virions might be masked due to the sludge particles surrounding the capsids, as suggested by Ward and Ashley (1979).

The decrease by ≥4 log10 of the faecal indicators is in agreement with results reported by Pereira-Neto et al. (1986) and Gantzer et al. (2001), who observed a decrease of 2 log10 to 5 log10 of the number of faecal indicators during composting of sludge in aerated static piles. The similar evolution of enterococci and E. coli during the composting, that we found, was also reported by Sesay et al. (1998), whereas other researchers have reported a longer survival of enterococci in relation to coliforms (Pereira-Neto et al. 1986; Deportes et al. 1998; Tiquia et al. 1998; Gantzer et al. 2001).

We observed that Salmonella was not isolated after the first month of composting whereas faecal indicators were detected even after the end of the maturation period. This is in agreement with results of Pereira-Neto et al. (1986), Droffner and Brinton (1995) and Tiquia et al. (1998) who have reported that Salmonella decayed more rapidly than faecal indicators. However, in the study presented here, the small numbers present in the initial product may also explain the absence of Salmonella and the limit of detection was rapidly achieved. Moreover, the low values of C/N ratio, ranged between 9 and 7, could have limited the bacterial multiplication as, according to Russ and Yanko (1981) and Yeager and Ward (1981), C/N ratios lower than 15 would involve an inactivation of Salmonella development.

In our study, the concentrations of Listeria sp. were positively correlated with those of enterococci and suggest a similar survival rate of the two types of bacteria which has also been reported by De Luca et al. (1998). Furthermore, given the similar threshold of detection for both Salmonella and Listeria (e.g. 0·12–0·15 MPN g−1 d.m.), the presence of L. monocytogenes after 4 months of composting suggests that L. monocytogenes have a greater survival potential during composting than Salmonella. This is in agreement with the data of Watkins and Sleath (1981) who reported a better survival of Listeria than Salmonella in soil having received sludge. The slow inactivation of L. monocytogenes during composting is in agreement with the data reported by Droffner and Brinton (1996), who artificially contaminated a compost constituted of leaves and food with this bacteria and have detected the presence of this pathogenic bacteria after 8 days of composting at 64°C. It is thus possible that the presence of L. monocytogenes until the fourth turning is due to a better environmental adaptation of this bacteria than Salmonella during the fermentation phase of composting.

In spite of a reduction of 4 log10, C. perfringens was still present in the mature compost at concentrations of 8 × 102 CFU g−1 d.m., which supports the hypothesis that natural ventilation of the compost does not involve the complete destruction of the spores of Clostridium, as was noted by Böhnel and Lube (2000). Indeed, this survival may be explained by anaerobic zones within the composting pile such as packing at the bottom resulting in areas less oxygenized than the other areas. The existence of those anaerobic zones made the growth of anaerobic bacteria possible.

For the enteric micro-organisms taken as a whole, no correlation could be established, in our study, between their decrease and any of the analysed physico-chemical parameters. Nevertheless, it is known that the dynamics of micro-organism populations are influenced by factors such as temperature, oxygen content, water content, pH (Epstein 1997) and by other factors not taken into account in this study such as competition and production of inhibiting substances by the indigenous microflora (Droffner and Brinton 1995; Sidhu et al. 2001). However, whereas the three areas, monitored individually, differed essentially for temperature, water content and pH (Table 3), no difference appeared in the decrease of micro-organism densities. One of the most important factors that affects enteric micro-organisms inactivation is the temperature. Thus, Sesay et al. (1998) observed a greater elimination of faecal indicators in a compost artificially ventilated, where the temperature exceeded 55°C for 20 days, than in naturally ventilated compost for which the temperature of 55°C was maintained for 11 days. In our study, while the temperature maxima of about 41°C recorded at the bottom was always lower than the temperature of the two other areas (ranging between 53 and 69°C), the numbers of the target micro-organisms were similar regardless the position of the pile. This is in agreement with the results obtained by Tiquia and Tam (2000) who have also distinguished the three zones to be studied in a pile of compost. In contrast, Pereira-Neto et al. (1986), working on static composting system, have reported that the top of the pile produced a greater reduction in number of faecal indicators than the middle and the bottom of the pile. In our experiment, the results could be explained by the monthly blending of the pile which homogenized the three areas, what does not permit a significant display of differences.

The increase in numbers of faecal indicators we observed after 1 month of composting (Fig. 3) has already been reported by Sesay et al. (1998), who observed, after each turning of compost, a regrowth of faecal indicators which they put down to a contamination of the mixture by the external zone of the compost nonaffected by the rise in temperature. This hypothesis could explain why, in our study, the bacterial densities, after the turning of the compost, were always higher than those obtained in each zone of the pile. Furthermore, Hay (1996) who studied the regrowth of Salmonella suggested that it was not only due to the effectiveness of the thermal inactivation but also to external factors such as blending of bulking agent and external sources of contamination. In our case, it is also possible that during the turning of the heap, which took 1 day, the compost has been contaminated by soil and dust or by the material used to blend the compost.

To summarize, the hygienic effect of sludge composting in mixture with straw results in a significant reduction of enteric micro-organisms. However, faecal indicators were not completely inactivated, as their concentration ranged between 64 and 102 bacteria g−1 d.m. in the final product. As reported by Sidhu et al. (2001), the technique of composting does not guarantee complete hygienization of the end product, insofar as it is necessary to take account of a potential regrowth of bacteria. In spite of a variability of results (from 1 to 2 log10) obtained on each series of four samples, a progressive inactivation of all target micro-organisms appeared during composting. In any case, our results show that the turned pile composting technique used in this study, well adapted to rural areas, where agricultural equipment may be used to handle them, allowed us to obtain the same hygienic effect obtained with the forced aerated pile.

One point that our work also shows has to be focused: use of faecal E. coli or enterococci as indicators of hygienization, which was proposed by Pereira-Neto et al. (1986), could be questioned, as the resistance of pathogenic bacteria differs from one pathogen to another, similar to that observed with Salmonella and L. monocytogenes.

Finally, the choice of mixing a small quantity of straw to the sludge had been made for economic reasons. This choice led to: (i) a high moisture level at the bottom of the pile and a too sticky characteristic of the matrix, resulting in weak air circulation and difficulty in handling the initial mixture or the compost during the first turnings; (ii) a low C/N ratio at the beginning of the composting, resulting in release of gaseous nitrogenous compounds. As a matter of fact, a better technico-economic budget and an improvement in the sought after effects of composting, notably the decay of sludge micro-organisms, by increasing temperature namely, can thus be obtained easily by an increase of the straw/sludge ratio.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the Regions Pays de la Loire and Bretagne. The authors thank the ‘Pôle agronomique Ouest’, with whom this research was initiated. The team of the C.A.T. 4 Vaulx concluded the operations relating to the handling of the heap of compost, and we are particularly grateful to J. Mazé for his participation and advice. We thank H. Yuliprianto, who helped us very much on the fringe of his own work, N. Nicholson, G. Mesnard and F. Rama-Heuzard for their technical assistance, and Y. Picard for having finalized Fig. 1. We also thank Angers Agglomération, who allowed us to take the sludge. We are indebted to IDAC Laboratory and to COOPAGRI-Bretagne and particularly to R. Roudaut and M. Lasbleiz, for measuring the analyses of physico-chemical parameters and lending us a press-truck.

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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