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Biodigestion of kitchen waste

A comparative evaluation of mesophilic and thermophilic biodigestion for the stabilisation and sanitisation of kitchen waste

Final report prepared by The University of Southampton and Greenfinch Ltd

Funded by BIFFAWARD and South Shropshire District Council through the Landfill Tax Credit Scheme

CONTENTS
Summary...4 1. Introduction...5 2. Aims and objectives..6 3. Methodology.....6 4. Results and discussion..8 4.1 Kitchen waste collection.8 4.2 Pre-processing of kitchen waste........11 4.3 Optimisation of mesophilic and thermophilic digestion...12 4.4 Pathogen control in mesophilic and thermophilic digestion.17 Conclusions.24 FIGURES Figure 1: The thermophilic and mesophilic digester systems, showing the mixed feed tanks, digester and collection tanks...7 Figure 2: Schematic diagram of the anaerobic digester system...8 Figure 3: Weight of kitchen waste collected from Burford village and Tenbury Wells..9 Figure 4: Average composition of source separated waste (by weight).10 Figure 5: Frequency of particle size (mm) of 100 samples of shredded raw kitchen waste, mixed feed and digestate.11 Figure 6: pH and alkalinity of the mesophilic and thermophilic digesters.13 Figure 7: Concentration of volatile fatty acids, biogas yield, percentage of methane and retention time in the (a) mesophilic and (b) thermophilic digesters..15 Figure 8: Concentration of Salmonella in the (a) mesophilic and (b) thermophilic anaerobic digester systems..19 Figure 9: Concentration of E. coli in the (a) mesophilic and (b) thermophilic anaerobic digester systems. .................20 Figure 10: Concentration of Entercocci in the (a) mesophilic and (b) thermophilic anaerobic digester systems. ....21 TABLES Table 1: The effect of hygiene measures on concentrations of Salmonella, E. coli and Enterococci in mesophilic and thermophilic digestate.23 References...25 APPENDICIES Appendix 1: Analytical methods26 Appendix 2: Lithium Tracer Test...32 Appendix 3: Kitchen Waste Collection......36 Appendix 4: Hygiene Regime........41

SUMMARY Aim of the project The aim of the project was to establish whether anaerobic digestion, possibly combined with heat treatment, is a safe means of processing kitchen waste and restaurant waste, to determine whether the disposal of the stabilised and sanitised digestate would meet the draft EU Animal By-Products Regulation. Methodology Kitchen waste was collected on a weekly basis from the village of Burford, Shropshire. The waste was shredded at the anaerobic digestion site and treated in two digesters, one operating at mesophilic temperature (36.5oC) and the other at thermophilic temperature (56oC). The digesters were operated on a six hour cycle and the volatile solids loading was varied to optimise the system. Samples were taken from the digesters, feedstock and collection tanks to assess (among others) biogas yield, volatile solids reduction, and presence of bacteria and pathogens. Main findings Source separation of the organic fraction of municipal waste yields on average 2.9 kg per household per week with less than 2.5% contamination. In all, 23.2 tonnes of kitchen waste was diverted from landfill. The average moisture content of the source-separated kitchen waste was 78.4%, and volatile solids were found to be 92.4%. Thermophilic digestion of kitchen was found to be less stable than mesophilic digestion. Mesophilic digestion medium has a greater buffering capacity and is more robust to changes or accumulation of inhibitory chemicals. The biogas in both systems comprised of 58% methane. The mesophilic system produced 164 m3 per tonne, whereas the thermophilic system produced 157m3 per tonne. Kitchen waste was collected on a weekly basis and contained pathogens such as Salmonella, Escherichia coli and Enterococci. Thus, this waste would require further treatment before it could be applied to agricultural land to meet the draft EU Regulations. Mesophilic digestion produced a 3.0 log reduction for E. coli, 2.8 log reduction of Enterococci and 1.4 log reduction of Salmonella. Overall, thermophilic digestion produced a 5.07 log reduction for E. coli, 4.0 log reduction of Enterococci and 2.2 log reduction of Salmonella. Digestate from the collection tanks in both the mesophilic and thermophilic systems still contained Salmonella, and would therefore require further treatment prior to spreading on agricultural land. The introduction of a hygiene regime reduced the presence of Salmonella, E. coli and Enterococci in thermophilic digestate. Laboratory-scale pasteurisation at 70C for at least 1 hour effectively eliminated Salmonella, E. coli and Enterococci. When the digestate is stored at 20C, Salmonella persists for 10 days and E.coli for 21 days, but Enterococci was still present after 3 months.

Introduction

Alternative processing technologies, such as composting or anaerobic digestion, offer some potential for recovery of value from organic wastes by producing soil-conditioning composts and, in the case of anaerobic digestion, biogas. Therefore, anaerobic digestion and composting are both being considered as possible routes for treatment of the biodegradable fraction of municipal solid wastes, and it is anticipated that their use will become more widespread over the coming years. In the UK, the cost of landfilling is rising rapidly and the gap between landfill disposal and alternative processing routes is narrowing. However, there is currently a reluctance to invest in anaerobic digestion systems. This is partly because anaerobic digestion is a new technology for solid waste recovery and also because there are uncertainties surrounding the potential beneficial use of the digestate produced. The agricultural/horticultural and retail gardening markets are reluctant to accept new products for which there are no required standards or prior experience in their use. In addition, there is uncertainty surrounding the safety of the product. In 2002, at the start of this research project, the Animal By-Products Order Amendment was in force in the UK. The Amendment, enforced after the outbreak of Foot and Mouth Disease, did not permit the products of composting and digestion to be spread on agricultural land, and therefore, the only disposal route was landfill, or an energy-from-waste plant. At the start of the project the EU Animal By-Products Regulation was still in draft form. The Draft Regulation stated that materials that are considered low risk (e.g. food wastes containing meat or those that have been in contact with meat) could be treated by composting or anaerobic digestion so long as processing included an in-vessel stage, the particle size of the material was reduced to less than 12 mm, the material was heated to 70oC for one hour, and pathogen limits for Salmonella and Enterobacteriaceae were met. Application to arable land of products from composting and anaerobic digestion would then be permitted. The Regulation would also permit Member States to adopt different approaches to treatment, so long as these were shown to achieve the pathogen limits. The EU draft recommendations were adopted in May 2003, (EU Regulation Laying Down Health Rules Concerning Animal By-Products Not Intended for Human Consumption (EC 1774/2002)) and are implemented in the UK by the Animal Byproducts Regulations (England) 2003. The UK Regulations includes alternative treatment standards, in which minimum time/temperature and particle size reduction specifications must be met. The alternatives include: for composting in a closed vessel reactor, a maximum particle size of 40cm treated with a minimum temperature 60oC for two days; for composting in a housed windrow, a maximum particle size of 40 cm, treated with a minimum temperature of 60oC for eight days during which the windrow must be turned three times at no less than two day intervals; for composting in a closed vessel or anaerobic digestion, a maximum particle size of 6 cm, treated with a minimum temperature of 70oC for one hour; and, for anaerobic digestion, a maximum particle size of 5 cm, treated with a minimum temperature of 57oC for five hours. 5

Approval for an anaerobic digestion plant will specify one of the above methods and, in addition, will stipulate that: measures are taken at source to ensure that meat is not included in the catering waste; or, that following treatment the material is stored for an average of 18 days after treatment (storage does not need to be in a closed system). As in the EU Regulation, the UK Regulation requires anaerobic digestion plant operators to test representative samples for Salmonella and Enterobacteriaceae, or where only catering waste is treated, for Salmonella only. 2 Aims and objectives

The objective of the project was to establish whether anaerobic digestion, possibly combined with heat treatment, is a safe means of processing kitchen waste and restaurant waste, to determine whether the disposal of the stabilised and sanitised digestate would meet the draft EU Animal By-Products Regulation. The aims of the project were: Optimisation of the anaerobic process for maximum energy production To investigate the reduction of pathogens after anaerobic digestion at 35C with a minimum hydraulic retention time of 14 days. Heat treatment will be given (at 60C for 3.5 hours or 70C for one hour) if necessary. To investigate the reduction of pathogens after anaerobic digestion at 55C with a minimum hydraulic retention time of 7days and with a guaranteed minimum residence time of 6 hours. Heat treatment will be given (at 60C for 3.5 hours or 70C for one hour) if necessary. To control odour during the mesophilic (35C) and thermophilic (55C) anaerobic digestion process. Methodology

Kitchen waste was collected on a weekly basis from a local village and a catering establishment close to the anaerobic digester site. The waste was shredded at the site and treated in two purpose-built 1500-litre anaerobic digesters (figure 1). Each digester system comprised a feed tank with macerator, the digester, and a digestate collection tank (figure 2). Under normal operation, the shredded kitchen waste was added to the feed tank together with recycled digestate taken from the collection tank. The mixed waste was macerated in the feed tank and then pumped to the digester on a six hour cycle. After digestion, the digestate was pumped to the collection tank. The design of the digesters was based on a full-scale anaerobic digester with its own internal heating system and gas mixing system to ensure a constant homogeneous mixture and temperature. Each digester system was completely independent, one operating at mesophilic temperature (36.5C) and the other at thermophilic temperature (56C). The temperatures were monitored throughout the experiment via thermocouples positioned at various heights within the digester. Samples were taken daily from the digesters for analysis of a number of process parameters such as: ammonia and volatile fatty acid concentrations, and pH. Biogas yield and the concentration of methane in the gas were also monitored daily. On a weekly basis, samples were taken from the shredded kitchen waste, the mixed feed, digester contents, and collected 6

digestate. These samples were analysed for presence of Salmonella, Escherichia coli and Enterococci. Salmonella was chosen because the presence or absence of this human pathogen determines whether a compost or digestion process meets the treatment standards proscribed by the draft EU Regulation. E. coli was measured because it is a common indicator of possible faecal contamination. Enterococci (a sub-grouip of the more general group of faecal indicator bacteria) is a resilient organism which survives thermal treatment after many pathogenic bacteria, viruses and parasite eggs are killed or lose their viability. The analytical methods used during the project can be found in Appendix 1. A Lithium tracer test was carried out to demonstrate the effectiveness of the mixing system of each digester. Further details of the method used and the results of the tracer test are given in Appendix 2.

Figure 1: The thermophilic and mesophilic anaerobic digester systems, showing the mixed feed tank, digester and collection tanks.

Shredded Kitchen waste Mixed feed tank Digester Digestate collection tank

Biogas

Figure 2: Schematic diagram of the anaerobic digester system.

4 4.1 4.1.1

Results and Discussion Kitchen waste collection Source separation

On a weekly basis, a kerbside collection of kitchen waste was operated in Burford Village and Tenbury Wells. The kitchen waste was placed in a plastic bag by the householder. On receipt at the digester site, the kitchen waste was inspected for contaminants (such as metals and plastics) which were removed. Source-separation of the organic fraction of household waste relies heavily on the co-operation and participation of the householder. The 14-month collection scheme reflected a high level of participation and co-operation by the householders. The scheme had over 90% participation rate reflecting the high level of support from the participants to this small-scale community project. Between 80 and 120 householders participated during the scheme. Initially only 80 householders were invited to participate to allow the digesters to adjust to the organic loading rates. Week 50 of the collection (19.07.02) saw the increase in householder participation due to a planned recruitment phase. Appendix 3 shows the recruitment leaflet sent to the householder and gives further details of the collection scheme. Only 1.5-2.5% of the sourceseparated kitchen waste was recorded as non-putrescible material. This figure also includes the plastic bag that was used to collect the waste. 4.1.2 Provision to the householder

Householders were supplied with a bin and plastic liners free of charge. The supply of plastic bags and a sealed bin appeared to encourage all-year participation to the scheme. There was no decline in source-separation during the summer months. However, it was noticeable that there was increased double-bagging and the waste was often partially degraded on receipt. Associated odours were heightened during the hot summer months, emphasising the need for a weekly collection of the waste during this period. Several householders commented that the separation of their waste meant that their black-bag waste didnt smell, leaving it cleaner and 8

less of a problem. Supply of the plastic bags was essential for this collection system to ensure that the householder participated in the scheme. Environmentally, the use of biodegradable bags would have been preferable, since the use of plastic bags would increase the waste material that would need to be incinerated or landfilled. However the cost of biodegradable bags precluded their use and the durability of these bags was questioned. It was also necessary to consider the potential for blockage of the anaerobic digester system, if biodegradable bags were shredded and added to the feedstock. 4.1.3 The collection system

The collection system was thorough and it was very rare that any complaints were received. During the entire research period, only 5 bags were missed. This was caused by new collection operators or the householder had put their kitchen waste bag out after the collection period finished. During the research period it became apparent that the vehicle and the collected waste must be well-ventilated due to the production of odours, and must be able to hold and retain organic liquid wastes. 4.1.4 Mass of Kitchen waste

During the 14-month project, 7,852 bags of source-separated kitchen waste were collected from the householders of Burford and Tenbury Wells. This amounted to 23.2 tonnes of kitchen waste. The collection started in Burford village, from which the total weight of the bags collected every week averaged 218.5 kg. After fifty weeks, householders in Tenbury Wells were brought into the scheme and the total collected weight rose to 350.1 kg per week, figure 3. Appendix 3 contains further details of the results from the kitchen waste collection.

Kitchen waste collected during anaerobic digestion study


10.0 500 no. of bags; weight of bags total (kg) 9.0 8.0 7.0 6.0 300 5.0 4.0 3.0 100 2.0
50% ncrease in participating households

200

average bag weight (kg)


Weight of Collected bags (kg) Total number of bags Average Bag weight (kg)

400

1.0 0.0

0 1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81

week

Figure 3: Weight of kitchen waste collected from Burford Village and Tenbury Wells.

The average weight of putrescible kitchen waste collected during the study was found to be 2.94 kg per bag, figure 3. There was a slight increase in the average bag weight when the householder numbers increased after week 51; the average bag weights rose from 2.87 kg for Burford village, to 2.95 kg for Burford and Tenbury Wells combined. The increase in average bag weight was not significant. In a previous study involving householders from a nearby town of Ludlow, the average bag weight was 4.2kg. The difference between the bag weights achieved in these two projects can be attributed to the type of participating householders. The Burford households are predominantly one or two persons, of which the majority can be classified as retired. The Ludlow participants, on the other hand, comprised a wider cross-section of society. The figure of 2.94 kg/household/week is very similar to recent Government statistics and results from other studies. Waste Not, Want Not (2002) reports that kitchen waste amounts to 17% of the total household waste, equivalent to 3 kg/household/kg. The results from Burford are also comparable to the total organic food waste generated by householders in a study based in Moray, Scotland, where an average of 2.91kg of organic kitchen waste per week was measured (Jones, 2002). 4.1.5 Composition of kitchen waste

The average composition of the kitchen waste source-separated by the householder was analysed on two occasions. A compositional analysis of eight bags (10% of the weekly collection) was carried out by weight (kilograms) figure 4. Appendix 3 contains the raw data collected. Over 60% of the average bag is composed of uncooked fruit and vegetable waste. Eggs and raw meat, the main pathogen sources, were found to be relatively low in mass at 1.3% and 1.4%, respectively; 12% was cooked meat.

Cheese 1% Eggs 1% Teabags 10%

Paper 1% Cooked fruit & vegetables 7%

Bread 7% Uncooked meat 1%

Cooked meat 12%

Uncooked fruit & vegetables 60%

Figure 4: Average composition of source separated waste (by weight). January, 2002.

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4.2

Pre-processing of kitchen waste

Shredded kitchen waste is added to the mixed feed tank, diluted with recycled feedstock and macerated. The aim of pre-shredding was to produce a consistent feed and reduced plant down-time due to pipe blockages by large organic objects. The maceration of the shredded kitchen waste improved mechanical action and digestibility. Although kitchen waste is a pliable material, it needs to be transformed into a more liquid form to allow the waste to be pumped. This was done by recirculation and maceration with liquid digestate or water added to reduce the solids content of the waste. The shredding procedure enabled easy removal of any plastic bags or cling-film from waste and produced a consistent feed within hours of adding the waste to the reception tanks. Shredding of the kitchen waste reduced its particle size and increased its surface area. The particle size of shredded, macerated waste and digested waste is shown in figure 5. It can be seen that most particles were less than 2mm thick, and none were greater than 12mm thick thus achieving the size reduction required by the Draft EU Regulation and the UK Animal Byproduct Order 2002 for alternative digestion technologies which require a maximum particle size of 5 cm or 6 mm (depending on the treatment temperature).

Particle size analysis of shredded waste, macerated waste and mesophilic digestate

80 60
%

Shredded Biowaste Macerated Biowaste Mesophilic Digestate

40 20 0
0 to 0.49 0.5 to 0.99 1.0 to 1.49 1.5 to 1.99 2.0 to 2.49 2.5 to 2.99 3.0 to 3.49 3.5 to 3.99 4.0 to 4.49 4.5 to 4.99 5.0 to 5.49 5.5 to 5.99 6.0 to 6.49 6.5 to 6.99 7.0 to 7.49 7.5 to 7.99 8.0 to 8.49 8.5 to 8.99 9.0 to 9.49 9.5 to 9.99

Particle size (mm)

Figure 5: Frequency of particle size (mm) of 100 samples of shredded raw kitchen waste, mixed feed and digestate.

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Reduction in particle size provides a greater surface area for the attachment of bacteria which promotes the hydrolysis step within the reception tank. Hydrolysis is performed by the excretion of extracellular enzymes (e.g. cellulase, protease, amylase, etc.) or other metabolic catalysts by the hydrolytic bacteria. The hydrolysis products (amino acids, sugars) are utilised by the microbes for the production of cell mass, intermediate products such as proprionic and butyric acids, other long chain fatty acids, and alcohols. These compounds are substrates for methanogenic bacteria which produce methane. The recycled digestate also acts as an inoculum. It introduces fermentative bacteria accelerating the breakdown of the food waste during the hydrolysis stage as described above. It was noted that the temperature in the reception tank increased (compared to ambient temperature), and the solubility of the feedstock increased because of microbial activity. However, the practice of recycling digestate eventually leads to an accumulation of inhibitory substrates, such as volatile fatty acids or ammonia, within the digester medium, and then water must be added to reduce the concentration of these chemicals. 4.3 Optimisation of Mesophilic and Thermophilic Digestion

4.3.1 Temperature Temperature has a pronounced effect on the removal of pathogens and the rate of gas production in anaerobic digestion. There are two temperature ranges associated with methane production: mesophilic (25 to 40C) and thermophilic (50 to 65C). The stability of thermophilic digestion is questionable, yet it is reported that there is an increased degradation of organic matter in the higher temperature range, leading to higher volatile solids destruction and higher biogas yield. However, thermophilic digestate may contain higher concentrations of ammonia-nitrogen than digestate from mesophilic anaerobic digestion. Each digester was fitted with a set of thermocouples to measure the temperature within the digestate. The average temperature of the thermophilic digester was 56.0oC (+/- 0.21oC); the average temperature for the mesophilic digester was 36.5oC (+/- 0.28oC). There was only slight variation between each thermocouple readout, showing that the mixing and heating systems were very effective at achieving a constant environment within the digester vessel, thus ensuring that the kitchen waste and associated bacteria were subjected to the same conditions. 4.3.2 pH

The mean pH values for mesophilic and thermophilic digesters were 7.54 (with a range of pH 7.30 to 7.78) and 7.48 (with a range of pH 6.82 to 7.81) respectively, figure 6. The average pH of the mixing tank was 4.5 in the mesophilic tank and 4.4 in the thermophilic tank. This initial pH resulted from the fermentation and hydrolysis phase of digestion. Both digesters became unstable during the study period. The pH of the mesophilic digester declined rapidly to 7.30 during week 38, and so the feedstock diluted by water. The same action was taken when the thermophilic digester pH plummeted to 6.82. In this instance, the digester feedstock was stopped for approximately a week to allow the complex bacterial population to recover and regain its balance. pH can be described as an indicator of the stability of the digester medium as it is dependent upon the buffering capacity of the digester itself. It is an indicator of the state of equilibrium of the complex system, which is influenced by number of chemical balances. pH is largely 12

dependent on CO2 concentration in the medium but it also influenced by volatile fatty acid and ammonia concentrations. However, the measurement of pH for the thermophilic and mesophilic digester mediums has been shown as a poor indicator of an unstable digester due to a delay in reaction time. This is caused by the buffering capability of the media and therefore pH cannot be monitored alone; it must be recorded alongside other parameters such as the volatile fatty acid (VFA) concentration and biogas composition.

Mesophilic Digester: pH and alkalinity


25000 8.00

Alkalinity (mg/l) Digester pH

7.50 20000 alkalinity (mg/l) 7.00

6.00 10000 5.50

5000 0 10 20
w eek

5.00 30 40 50 60

(a)
Thermophilic digester: pH and alkalinity
25,000 8.00 7.50 20,000 alkalinity (mg/l) 7.00 15,000 6.50 6.00 10,000 5.50 5,000
0 10 20 30 week 40 50 60 Alkalinity (mg/l) Digester pH

5.00

(b) Figure 6: pH and alkalinity of the (a) mesophilic and (b) thermophilic digesters.

pH

pH

15000

6.50

13

4.3.3 Alkalinity Alkalinity is effectively the acid-neutralising capacity of the medium which resists pH change caused by the accumulation of acids within the medium. Alkalinity results from the presence of hydroxides, carbonates, bicarbonates of calcium, magnesium, sodium potassium and/or ammonia in the digestate. VFA can also contribute to alkalinity. Both the mesophilic and thermophilic digesters had high alkalinity concentrations at 13,000 mg l-1 (range 9,250 to 19,125 mg l-1) and 16,400 mg l-1 (range 9,625-27,000 mg l-1) respectively, figure 6. There was much larger variation in thermophilic total alkalinity, reflecting the greater instability of the thermophilic system. The alkalinity concentrations found in the mesophilic and thermophilic systems were much higher than usually described in anaerobic digesters (typically 2000-4000 mg l-1). This may be due to the high nitrogen content of the kitchen waste feedstock (see 4.3.5). 4.3.4 Volatile Fatty Acids

Concentrations of total VFA as high as 45,000 mg l-1 (range 14,131-44,625 mg l-1) were recorded in the thermophilic digester; the mesophilic medium recorded a maximum VFA concentration of 28,000mg l-1 (range 6,825-28,263mg l-1), figure 7. The high standard deviation of the thermophilic VFA concentration (9127 mg l-1) indicated the greater fluctuations in the thermophilic digester; this digester was much more unstable than the mesophilic digester. It was observed that the volatile fatty acid concentration was a rapid indicator of the digesters stability. Elevated VFA concentration indicated that the digester medium was unstable. It was apparent that as the total VFA concentration rose, the microbial populations ability to utilise these compounds effectively was inhibited. The levels of acetic acid and proprionic acids appeared to be the VFA species that accumulated and started to cause an imbalance in the digester medium of each digester. The relationship between proprionic acid and instability was primarily seen in the thermophilic digester but also occurred in the mesophilic media. VFA accumulation has an inhibitory effect on methogenic bacteria and can cause depression in pH if the digester medium becomes unbalanced and is no longer able to buffer the concentration of VFA.

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(a)
Mesophilic Biodigester (Weekly 11/01/02 -21/02/03)
Biogas - m3/wk Retention Time - d CH4 - % VFA g/l

120 110

45 40 35 30 25 20 15 10 5 0
1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58

Biogas (m3/week) Methane (%) Retention time (days)

100 90 80 70 60 50 40 30 20 10 0

week

(b)
Thermophilic Biodigester (Weekly 11/01/02 -21/02/03 )
Biogas - m3/wk Retention Time - d CH4 - % VFA g/l

120 Biogas (m3/week) Methane (%) Retention Time (days) 110 100 90 80 70 60 50 40 30 20 10 0 1 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 week

45 40 35 30 25 20 15 10 5 0 VFA (g/l)

Figure 7: Concentration of volatile fatty acids, biogas yield, percentage of methane and retention time in the (a) mesophilic and (b) thermophilic digesters.

VFA (g/l)

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4.3.5

C:N ratio

Carbon and nitrogen are essential for cell synthesis and metabolism of anaerobic digestion and the carbon to nitrogen ratio of a substrate can affect the digestion process. The carbon to nitrogen ratio of the digester feedstock (for both digesters) was 9:1. This ratio is distinctly different to the often documented optimum C:N ratio of 25 to 30: 1. During the digestion phase, the carbon is utilised to produce CO2 and CH4, leading to the reduction in carbon content and the C:N ratio decreased to 3:1. This indicates a level of carbon deficiency that is the result of high nitrogen content of the kitchen waste and the recirculated digestate. 4.3.6 Total and volatile solids

The calculation of the total solids and volatile solids percentage of a feedstock and the digester process can confirm the conversion performance of the digester (organic loading rate and the biogas production). The source separated kitchen waste was shown to have a dry solids content of 21.6% (with a range between 16.2 to 27.3%) and a volatile solids content of 92.4%. The thermophilic and mesophilic feedstocks contain, on average, 54% and 50% of fresh shredded kitchen waste respectively. The remainder of the feedstock was made up of recirculated digestate and/or water to reduce the total solids content. The mean total solids of the thermophilic feedstock was 12.2%, with a volatile solids content of 89.1%; the mean total solids of the mesophilic feedstock was 12.23% with a volatile solids content of 89.1%. The total solids content reduced to approximately 5% for both digesters, and the volatile solids content was approximately 88%. 4.3.7 Loading Rate

The mesophilic digester was started at a mean retention time of 31.5 days, with a specific volatile solids loading rate of 4.1 kg VS m-3d-1. The retention time was reduced over a number of weeks to 20 days (specific loading rate of 5.72 kg VS m-3d-1) but this loading rate was found to be too high, causing accumulation of VFAs and pH depression. The thermophilic digester process was started at a longer mean retention time of 44.5 days, with a specific volatile solids loading rate of 3.01 kg VS m-3d-1. The thermophilic digesters maximum specific VS loading rate was 5.47 kg VS m-3d-1, however this rate was not sustainable the thermophilic digester could not tolerate an organic loading rate equivalent to the mesophilic digester. The possible limiting factors are not completely clear, however it can be assumed that the accumulation of inhibitory substances and the inability of thermophilic bacteria to function efficiently caused the maximum VS loading rate to be unsustainable. The thermophilic digesters performance could be considered poor in comparison to the performance of other thermophilic systems. For example, in a one-stage wet system treating OFMSW and other biowastes, the maximum organic loading rate was found to be 9.7 kg VS m-3d-1 when fed mechanically sorted OFMSW (Pavan et al, 1994). However, when the feed in this digester was altered to source-separated biowaste, the maximum organic loading rate reduced to 6 kg VS m-3d-1, a similar value to this experiment. However, the thermophilic digester produced a higher biogas yield per VS loading. Although the average biogas yield for the thermophilic digestion process was 690 l kg-1 VS fed, its maximum conversion rate was 1,060 l kg-1 VS. Mesophilic digestion produced a higher 16

average of 716 l kg-1 VS but its maximum was only 882 l kg-1 VS fed. This suggests that if optimum conditions could be sustained, the thermophilic digestion process could produce a higher biogas yield than the mesophilic system. The thermophilic digestate production was 83% by weight of the incoming feedstock (kitchen waste) and its dry matter was 5.5% whereas mesophilic digestate production was 90% by weight of the incoming feedstock (kitchen waste) and its dry matter was 5.4%. The average volatile solids reduction of the feedstock during the mesophilic digestion process was 67% with a maximum reduction of 82.6%. The thermophilic process achieved an average volatile solids reduction of 70%, with an maximum of 82%. 4.3.8 Biogas production and composition Biogas production and composition are influenced by the accumulation of inhibitory chemicals such as volatile fatty acids and the organic loading rate. The biogas composition and production was measured daily. It was found that the average methane composition for mesophilic digestion was 57.9% (maximum 64.2%; minimum 42.1%) and for thermophilic digestion was 57.6% (maximum 65.8%; minimum 49.8%). Trace gases were present in the biogas at relatively low levels. Mesophilic biogas contained traces of hydrogen sulphide (1,300 to 1,700ppm) and carbon monoxide (200 to 500ppm). The thermophilic biogas contained similar concentrations of hydrogen sulphide (1,500 to 2,500ppm) and carbon monoxide (290 to 700ppm). It was calculated that the mesophilic digester transformed a kilogram of kitchen waste into 164 litres of biogas with an optimum gas production of 271 litres biogas kg-1 kitchen waste. Similarly, the thermophilic digester transformed one kilogram of kitchen waste into 157 litres of biogas. The optimum figure obtained was 274 l biogas kg-1 kitchen waste. 4.4 Pathogen control in Mesophilic and Themophilic Digestion

Operational parameters such as pH, alkalinity, VFA and retention time affect the survival of bacterial pathogens. Temperature has the most impact on pathogen reduction, although it has been shown that pH can influence pathogen inactivation. Figures 8 to 10 show the concentrations of Salmonella, E. coli and Enterococci in kitchen waste, mixed feed, digester contents and collected digestate for the thermophilic and mesophilic digester contents. 4.4.1 Kitchen Waste

Salmonella was present in all samples (25g) of raw kitchen waste, with a median concentration of 46 MPN/g (wet weight). The concentration ranged from 15 to 450 MPN/g during the study period. Thus, the kitchen waste collected in this study could not be applied to agricultural land without further treatment to meet the draft EU Regulations. Escherichia coli (median 7.85 x 105 cfu/g wet weight) and Enterococci (1.01 x 106 cfu/gram wet weight) were also found. Similarly, Bohm and co-workers (1999) found Salmonella, E. coli and faecal streptococci in biological and household waste, in addition to other bacteria, such as Clostridia, Enterobacter, Yersinia and Staphylococci. There was no trend in concentrations of these bacteria during the year. The concentrations of the E. coli and Salmonella were judged to be quite high, compared with values reported for food waste in the literature. It is possible that the values were influenced by the analytical 17

techniques used. In addition, the waste spent up to seven days in a bin prior to collection; this might encourage the growth in numbers of bacteria prior to analysis, especially if kept indoors at room temperature.

4.4.2 Mixed feed tanks The majority of samples from the mesophilic mixed feed tank on day one (after the kitchen waste was mixed with mesophilic digester liquor) showed the presence of Salmonella, but at lower concentrations than in the kitchen waste averaging 18.5 MPN/g wet weight. E. coli (9.23 x 104 cfu/g) and Enterococci (1.34 x 105 cfu/g) had decreased by one order of magnitude compared to the kitchen waste. After seven days in the mixed-feed tank, Salmonella were absent or at the limit of detection of the MPN method (0.04 MPN/g). There was a further order of magnitude decrease in the concentrations of E. coli (6.17 x 103 cfu/g) and Enterococci (6.23 x 103 cfu/g). In the thermophilic digester system, Salmonella was detected in only half of the 25g samples taken from the feed tank on day one, and after seven days only two samples showed the presence of the pathogen. The concentration of Salmonella in the feed tank was close to the detection limit of the analytical technique. The concentrations of E. coli (2.27 x 104 cfu/g) and Enterococci (1.66 x 104 cfu/g) in the feed on day one had decreased by nearly two orders of magnitude compared to the initial kitchen waste. By the end of the week a further two orders of magnitude decrease was measured; the concentration of E. coli had declined to 4.2 x 102 cfu/g, and that of Enterococci had declined to 5.6 x 102 cfu/g. The pH of the mixed-feed was 4.4 due to the presence of high concentrations of unbuffered volatile fatty acids and the hydrolysis reactions taking place in the medium. Pathogenic bacteria can survive acidic conditions for a short time, but it is likely that they would be damaged or eradicated by seven days in the mixed feed tank at pH 4.4. For example, it is known that Salmonella can survive low pH environments (Foster and Spector, 1995). Fukushi et al (2003) observed that Salmonella survived in digested sewage sludge for two days when the pH was less than 5.0. The greater decrease in bacterial concentrations in the thermophilic mixed feed may be due to the presence of higher concentrations of volatile fatty acids from the recycled digestate, and the higher concentration of proprionic acid found in the thermophilic digestate.

18

Mesophilic digester system: Salmonella


1.00E+08 1.00E+07 1.00E+06

Salmonella (MPN/g)

1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 1.00E-01 1.00E-02


Kitchen w aste Mixed feed Mixed feed Digester day 1 day 7 contents Digestate in collection tank

week 24 week 26 week 28 week 30 week 33 week 36 week 38 week 42 week 48 week 52

week 25 week 27 week 29 week 32 week 34 week 37 week 41 week 46 week 50 week 55

(a)

Thermophilic digester system: Salmonella


1.00E+08 1.00E+07 1.00E+06

Salmonella (MPN/g)

1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 1.00E-01 1.00E-02


Kitchen w aste Mixed feed day 1 Mixed feed day 7 Digester contents Digestate in collection tank

week 24 week 26 week 28 week 30 week 33 week 36 week 38 week 42 week 48 week 52

week 25 week 27 week 29 week 32 week 34 week 37 week 41 week 46 week 50 week 55

(b) Figure 8: Concentration of Salmonella in the (a) mesophilic and (b) thermophilic anaerobic digester systems.

19

Mesophilic digester system: E.coli (cfu/g)


1.00E+08 1.00E+07 1.00E+06

w eek 16 w eek 19 w eek 22 w eek 24 w eek 26 w eek 28 w eek 30 w eek 33 w eek 37 w eek 39 w eek 42 w eek 48 w eek 52 Kitchen w aste Mixed feed day 1 Mixed feed day 7 Digester contents Digestate in collection tank

w eek 18 w eek 20 w eek 23 w eek 25 w eek 27 w eek 29 w eek 32 w eek 36 w eek 38 w eek 41 w eek 46 w eek 50 w eek 55

E.coli (cfu/g)

1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00

(a)

Thermophilic digester: E. coli (cfu/g)


1.00E+08 1.00E+07 1.00E+06 week 16 week 18 week 20 week 23 week 25 week 27 week 29 week 32 week 36 week 38 week 41 week 46 week 50 week 55 1.00E+05 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00
Kitchen w aste Mixed feed day 1 Mixed feed Digester day 7 contents Digestate in collection tank

E. coli (cfu/g)

week 19 week 22 week 24 week 26 week 28 week 30 week 33 week 37 week 39 week 42 week 48 week 52

(b)

Figure 9: Concentration of E. coli in the (a) mesophilic and (b) thermophilic anaerobic digester systems.

20

Mesophilic digester system: Enterococci


1.E+08

1.E+07

week 16 week 18 week 20 week 23 week 25 week 27 week 29

week 17 week 19 week 22 week 24 week 26 week 28 week 30 week 33 week 37 week 39 week 42 week 48

1.E+06

Enterococci (cfu/g)

1.E+05

1.E+04 1.E+03

week 32 week 36 week 38 week 41

1.E+02

1.E+01

week 46 week 52
Kitchen w aste Mixed feed day 1 Mixed feed day 7 Digester Digestate in contents collection tank

1.E+00

(a)
Thermophilic digestion system: Enterococci
1.00E+08

1.00E+07

week 16
1.00E+06

week 17 week 19 week 22 week 24 week 26 week 28 week 30 week 33 week 37 week 39 week 42 week 48 week 52

week 18 week 20 week 23 week 25 week 27 week 29 week 32 week 36 week 38 week 41 week 46 week 50
Kitchen w aste Mixed feed day 1 Mixed feed Digester day 7 contents Digestate in collection tank

Enterococci (cfu/g)

1.00E+05

1.00E+04

1.00E+03

1.00E+02

1.00E+01

1.00E+00

(b) Figure 10: Concentration of Enterococci in the (a) mesophilic and (b) thermophilic anaerobic digester systems.

21

4.4.3 Mesophilic Digester During the course of the study samples of the mesophilic digester contents were assessed for the presence or absence of Salmonella in 25g samples. Salmonella was detected in the majority of the digester samples, and the concentration averaged 4.5 MPN/g. The results demonstrated that the mesophilic digester system could achieve a 1.4 log reduction in Salmonella between the raw kitchen waste and the digestate in the digester. The mesophilic system achieved a 3.0 log reduction for Escherichia coli (from 9.1 x 105 to 9.2 x 102cfu/g); there was a 2.8 log reduction of Enterococci (from 1.9 x 106 to 4.1 x 103 cfu/g). The presence of Salmonella in the samples of mesophilic digestate mean that it could not be spread on agricultural land without further treatment to eliminate the pathogen. Salmonella was found to be present in the digester during weeks when the pathogen was not culturable in the samples of seven-day old mixed waste. This suggests that the pathogens present in the digester originated from bacteria which were viable in the feed earlier in the week, or that the digester sample had somehow been contaminated with raw kitchen waste. The results from the mixed feed samples and digester samples appear to support Kuntes (1998) suggestion that a twostage digestion system (acidophilic followed by methanogenic) could eliminate Salmonella, so long as adequate time is spent in the acidophilic stage. In this study, it would appear that the mesophilic digester would not be contaminated with Salmonella if it were supplied with mixed feed that was over seven days old. Since pathogens and indicator bacteria were recovered from the mesophilic digester, it appears that these organisms are able to survive at 37oC with high concentrations of VFAs at approximately neutral pH for at least six hours (the time between pumping out of the digester), and potentially for a longer period of time. Other research has demonstrated that pathogens can survive for long periods in extreme environments. In batch laboratory experiments, Kunte (1998) showed that Salmonella persisted for twelve days with a total VFA concentration of 5000 mg/l and pH 5.9 to 6.1, although the initial concentration of Salmonella was much higher (107 cells/ml) than in the current project. Fukushi et al, (2003) demonstrated that Salmonella could survive for nine days with a VFA concentration of 7500 mg/l and pH between 5.5 and 7. The VFA concentrations in the mesophilic digester were much higher in the current study, but the pH in the digester remained close to neutral because of high alkalinity. To test whether the kitchen waste and digestion samples were being contaminated by poor handling techniques, a hygiene regime was introduced at the plant in week 29. Table 1 shows that the concentration of Salmonella, E. coli or Enterococci in mesophilic digester samples was actually slightly higher after the introduction of hygiene measures, although the rise was not statistically significant. It was surmised that the presence of these indicator bacteria in the samples was caused by the low treatment temperature rather than contamination problems.

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Samples

Unit

Kitchen Waste

Mesophilic Digestate Prior to hygiene measures 4.01 879 4043

MPN/g Salmonella cfu/g E. coli Enterococci cfu/g

120 (15-450) 9.12 x 105 1.91 x 106

Mesophilic Digestate After introduction of hygiene measures 5.05 954 4315

Thermophilic Digestate Prior to hygiene measures 2.1 15 227

Thermophilic Digestate After introduction of hygiene measures 0.14 3 162

Table 1: The effect of hygiene measures on concentrations of Salmonella, E. coli and Enterococci in mesophilic and thermophilic digestate.

4.4.4 Thermophilic Digester At the start of analysis, Salmonella was present in three out of four of the 25 g samples taken from the thermophilic digester, and the average concentration was 2 MPN/g wet weight. E. coli was present at a concentration of 15 cfu/g and Enterococci were found at a concentration of 227 cfu/g. These concentrations are considerably lower than the concentrations found in the mesophilic digester. The increase in elimination in bacteria is explained by the high temperature encountered in the thermophilic digester, but may also be caused by the increased concentration of VFAs in this digester. After the introduction of the hygiene regime at the digester site, Salmonella was detectable in only 3 out of 15 weekly samples of thermophilic digestate. The concentration of Salmonella in the three contaminated samples ranged from 0.1 to 1.2 MPN/g wet weight. E. coli and Enterocococci were found more often and at concentrations of 3 cfu/g and 168 cfu/g, respectively. Thus, is appeared that the introduction of hygiene measures decreased the concentration of indicator bacteria found in digestate samples, however, the reduction in bacterial concentrations was not statistically significant. The thermophilic digester system demonstrated that, combined with the hygiene regime, a 2.2 log reduction in Salmonella numbers could be achieved between the input raw kitchen waste and the digestate. Similarly, thermophilic digestion achieved a 5.1 log reduction for E. coli, and a 4.0 log reduction for Enterococci. 4.4.5 Collection Tanks Samples taken from the mesophilic digester collection tank contained Salmonella, E. coli and Enterococci with the mean concentrations of 2.4 MPN/g, 1.3 x 103 cfu/g, and 2.3 x 104 cfu/g, respectively; mean concentrations in the collection tank from the thermophilic digester for Salmonella, E. coli and Enterococci were 2 MPN/g, 19 cfu/g and 3.7 x 103 cfu/g, respectively. In general, the concentration of E. coli in the collection tank was double that of the digester, while the concentration of Enterococci increased by an order of magnitude. It was thought that this might be a result of the reduced temperature of the digestate in the collection tank (i.e. ambient temperature), and the lower ammonia and volatile fatty acid concentrations found in 23

these open tanks. However, it was also possible that the digestate in the collection tanks was being contaminated by poor hygiene at the site, and after the introduction of hygiene measures, the collection tanks were cleaned out. This treatment did not have a significant impact on the numbers of pathogens or indicator bacteria recovered from the collection tanks. 4.4.6 Heat treatment Laboratory-scale pasteurisation at 70C for one hour effectively eradicates Salmonella, E. coli and Enterococci. This meets the standards prescribed in the draft EU Regulation. It was found that heat treatment at 57C for five hours eliminated Salmonella and E. coli but Enterococci were still recovered. 4.4.7 Storage Samples of mesophilic digestate were stored at 20oC for 3 months. Salmonella persisted for 10 days, whereas E. coli lasted for 21 days. Enterococci, however, were still recovered in the samples at the end of this period. Thus, if the mesophilic digestate was stored for 18 days, as required by the UK Animal By-products Order 2003, it would meet the treatment standard for Salmonella. Other research involving anaerobic digestion of cattle slurry has shown that some Salmonella species may survive nine days at mesophilic temperatures, and faecal streptococci may last over twelve days (Bohm et al, 1999). 5 Conclusions

Source separation of the organic fraction of municipal waste yields on average 2.9 kg per household per week with less than 2.5% contamination. In all, 23.2 tonnes of kitchen waste was diverted from landfill. The average moisture content of the source-separated kitchen waste was 78.4%, and volatile solids were found to be 92.4%. Thermophilic digestion of kitchen was found to be less stable than mesophilic digestion. Mesophilic digestion medium has a greater buffering capacity and is more robust to changes or accumulation of inhibitory chemicals. The biogas in both systems comprised of 58% methane. The mesophilic system produced 164 m3 per tonne, whereas the thermophilic system produced 157m3 per tonne. Kitchen waste was collected on a weekly basis and contained pathogens such as Salmonella, Escherichia coli and Enterococci. Thus, this waste would require further treatment before it could be applied to agricultural land to meet the draft EU Regulations. Mesophilic digestion produced a 3.0 log reduction for E. coli, 2.8 log reduction of Enterococci and 1.4 log reduction of Salmonella. Overall, thermophilic digestion produced a 5.07 log reduction for E. coli, 4.0 log reduction of Enterococci and 2.2 log reduction of Salmonella. Digestate from the collection tanks in both the mesophilic and thermophilic systems still contained Salmonella, and would therefore require further treatment prior to spreading on agricultural land. Laboratory-scale pasteurisation at 70C for at least 1 hour effectively eliminated Salmonella, E. coli and Enterococci. When the digestate is stored at 20C, Salmonella persists for 10 days and E.coli for 21 days, but Enterococci is still present after 3 months. The introduction of a hygiene regime reduced the presence of Salmonella, E. coli and Enterococci in thermophilic digestate.

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REFERENCES Animal By-products Regulations (England) 2003. Statutory Instrument 2003 No. 1482 ISBN 0110466748. HMSO, London. Bohm R., Martens, W. and Philipp, W. (1999) Regulations in Germany and results of investigations concerning hygienic safety of processing biowastes in biogas plants. In: Proceedings of the IEA workshop: Hygienic and environmental aspects of anaerobic digestion: legislation and experiences in Europe. pp 48-64 Univesitat Hohenheim, Stuttgart, Germany. ENDS Daily (1998). EU Parliament votes to strengthen landfill law. 20 February, 1998. (EU Regulation Laying Down Health Rules Concerning Animal By-Products Not Intended for Human Consumption (EC 1774/2002)) Fish, C.M., (1999). Enhancement of the anaerobic digestion of the organic fraction of municipal solid waste through nutrient supplementation. Ph.D Thesis. University of Southampton. Foster, J.W. and Spector, M.P., (1995). How Salmonella survive against the odds. Annual Review of Microbiolog. Vol 49, pp 145-174. Fukushi, K., Babel, S., and Burakrai, S., (2003). Survival of Salmonella spp. in a simulated acid phase anaerobic digester treating sewage sludge. Bioresource Technology, Vol 86, pp 177181. Kayhanian, M., (1995). Biodegradability of the organic fraction of municipal solid waste in a high-solids anaerobic digester. Waste Management and Research, 13, 123-136. Kunte, D.P., Yeole, J., Slonczewshi, J.l. and Foster, J.w., (1998). Inactivation of Salmonella typhi by high levels of volatile fatty acids during anaerobic digestion. Journal of Applied Microbiology, Vol 84., pp 138-142. Pavan P., Musacco, A., Cecchi, F., Gassetti, A. and Mata-Alvarez, J., (1994). Thermophilic semi-dry anaerobic digestion process of the organic fraction of municipal solid waste during transient conditions. Environmental Technology, 15, 1173-1182. Waste Not, Want Not - A strategy for tackling the waste problem in England (2002). Strategy Unit, Cabinet Office, London.

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Appendix 1: Analytical Methods 1. Daily monitoring Various physical and chemical parameters are monitored to check the status of the digester. Monitoring of the plant was carried out every day between 08.00 and 10.00 hours. Readings were taken to record gas production, biogas composition, digester temperature, retention time, discharge rate, and feed rate. 2. Physico-chemical monitoring 2.1. Temperature A 30-channel datalogger (DT500) recorded the output from thermocouples used to measure ambient temperature, and the temperature in the digester, feed tank and collection tank. Ten thermocouples were positioned in the digester at different levels within the vessel and temperature was recorded every 10 minutes. 2.2. Total solids The sample (approximately 10 g) was placed on a foil plate and dried to a constant weight at 105oC. Total solids % = Final weight (g) x 100/ Initial weight (g) 2.3. Volatile solids The dried residue from total solids analysis was weighed and heated in a crucible for two hours at 500oC in a preheated furnace. After cooling the crucible and ash were weighed. Volatile solids % = 100 ((V3-V1)100/(V2-V1)) Where V1 = weight of the crucible V2 = weight of dry residue and crucible V3 = weight of ash and crucible (after cooling) 2.4. pH Samples were analysed at ambient temperature with a Hanna Instruments temperature compensating pH meter. The meter was calibrated every day. Analysis was carried out immediately after sampling to avoid loss of carbon dioxide from the sample. 2.5. Volatile fatty acid and Alkalinity A standard method was adapted for on-site measurement of total volatile fatty acids and alkalinity. A 100 ml sample of digestate was collected in a beaker. The sample was filtered (Whatman No.2 filter paper) and the filtrate collected in a conical flask. The pH of the filtrate was noted. 20ml of filtrate was measured into a conical flask. 0.1M Hydrochloric acid was added from a burette until the filtrate is acidified to pH 4. The amount of hydrochloric acid used was noted. The filtrate was then heated and boiled for three minutes. After cooling, the filtrate was titrated with 0.01 M sodium hydroxide. The amount of sodium hydroxide used to reach pH 4 was noted, then the titration was continued to pH 7 and again the amount of sodium hydroxide was noted. The total volatile fatty acid content in mg/l acetic acid:

26

Acetic acid (mg/l) = (Volume of sodium hydroxide titrated from pH 4 to pH 7) x 87.5 2.6. Ammonia and other nutrients The concentration of ammonia and other nutrients such as nitrate, phosphate and potassium were measured using a Dr. Lange test kit. 3. Sample preparation for microbial analysis Samples from the kitchen waste and digester systems were taken in sterile containers every Friday. The samples were couriered to the laboratory within 24 hours of sampling. The following samples were taken: Fresh shredded kitchen waste - two random samples placed in two 300 ml containers. Seven day old reception tank waste 1000 ml sample taken from the reception tank before any new kitchen waste or recirculated digestate is added. Mix sample and decant 100 ml into a 100 ml sterile vessel. Repeat for the mesophilic and thermophilic digester streams. Fresh reception tank waste 1000 ml sample taken after all new kitchen waste and recirculated digestate has been added. The feed tank was well-mixed before the sample is taken. Decant 100 ml sample into a 100 sterile container. Repeat for the mesophilic and thermophilic digester streams. Digestate 1000 ml sample taken from the digester tank while gas mixing. Mix and decant 100 ml into a sterile 100 ml container. Return remaining digestate to the digester. Repeat for the mesophilic and thermophilic digester streams. Digestate Collection tank the tank was stirred to create a homogenous solution. 1000 ml sample was taken and mixed. 100 ml sample was decanted into a 100 ml sterile container. Repeat for the mesophilic and thermophilic digester streams.

4. Microbial analysis Salmonella 4.1. Introduction The procedure used is the British Standards Method Microbiology of food and feeding stuffs horizontal method for the detection of Salmonella (BS EN ISO 6579:2002). In addition to using the method for detection of Salmonella, the standard method was modified to enable counts of Salmonella to be made by the MPN technique. 4.2. Definitions Salmonella are defined as microorganisms which form typical or less typical colonies on solid selective media, and which display the biochemical and serological characteristics when tests are carried out in accordance with the BS standard method. The detection of Salmonella necessitates four successive stages: Pre-enrichment, Enrichment, Plating out, and Identification.

27

4.3. Apparatus 300 and 500 ml glass bottles with screw tops 20 ml universal bottles with screw tops Food blender 50 and 10 ml measuring cylinders 10 ml and 1 ml serological pipettes 90 Petri dishes Disposable loops 4.4. Media Buffered peptone water Salmonella enrichment broth (Rappaport and Vassiliadis). Double strength broth may be needed to ensure that the digestate and mixed feed achieve the correct pH of 5.2. XLD agar - dispensed into large petri dishes. Allow to dry before use. 4.5. Detection of Salmonella in 25 g samples. To 225 ml buffered peptone water, add 25 g of kitchen waste. The pH of acidic or acidifying foodstuffs is more stable if double strength buffered peptone water is used. Mix in a sterile food blender until homogenous. Between samples, was the food mixer goblet and rinse, then add boiling water and stand for five minutes. Pour off the boiling water and allow to cool. Incubate the buffered peptone water suspension for 18 hours at 37oC +/- 1oC. Transfer 1 ml of the pre-enrichment culture to a universal bottle containing 9 ml of double strength RVS broth. Incubate the inoculated RVS broth at 41.5 +/- 1oC for 24 +/- 3 hours. After incubation in the RVS broth, inoculate by means of a loop, the surface of one large size petri dish containing the selective plating out medium XLD agar. Typical colonies of Salmonella on XLD agar have a black centre and lightly transparent zone of reddish colour due to the colour change of the indicator. Salmonell H2S negative variants are pink with a darker pink centre. Lactose positive Salmonella on XLD agar are yellow with or without blackening. To confirm Salmonella, streak typical or suspect colonies onto the surface of pre-dried nutrient agar plates in order to obtain well isolated colonies. Incubate the plates at 37oC for 24 +/- 3 hours. Serological confirmation is carried out using a Microgen Salmonella kit. Results are expressed as presence or absence of Salmonella in 25 g of product. 4.6. Enumeration of Salmonella For enumeration of Salmonella, the pre-enrichment incubation in buffered peptone water is omitted. To 450 ml of quarter strength Ringers solution, add 50 g of sample. Mix in a food blender until homogenous. Wash and rinse the mixer goblet between samples. Use boiling water to sterilise the goblet. From the kitchen waste suspension, take three 1 ml samples from serial dilutions of 10-1 to 10-5 and add to 9 ml RVS broth in universal bottle. With the mixed feed and digestate samples, transfer the following volumes to universal bottles as follows: 100 ml culture + 100 ml double strength RVS broth 10 ml culture + 10 ml double strength RVS broth 1 ml culture + 9 ml double strength broth Repeat to produce 3 tubes for enumeration by MPN. Incubate the inoculated RVS broth at 41.5 +/- 1oC for 24 +/- 3 hours. Adjust the pH to 5.2 where necessary. Continue as for identification of Salmonella as described above. From the 28

results of serological examination, the concentration of Salmonella, in terms of MPN/g of kitchen waste, mixed feed or digestate is calculated. 5. Microbial analysis Escherichia coli 5.1. Introduction The method was adapted from ISO 16649-1:2001, Microbiology of food and animal feeding stuffs. Horizontal method for the enumeration of B-D-glucoronidase-positive Escherichia coli. Colony count technique at 44oC and 5-bromo-4-indolyl-B-Dglucoronide. The procedure uses a memebrane filtration technique using Membrane Lauryl Sulphate broth, and TBX agar (comprising a tryptone bile agar with a chromogenic substrate, 5-bromo-4-indolyl-B-D-glucoronide. 5.2. Definitions Organisms that are oxidase negative, produce acid from lactose and form all shades and sizes of yellow colonies on membrane filters (after incubation at 30oC for 4 hours followed by 37oC for 14 hours) are regarded as coliforms. Organisms which fulfil the above criteria after incubation at 30oC for 4 hours followed by 44oC for 14 hours are regarded as E. coli. Most strains of E. coli produce B-D-glucoronidase. Indoxyl released from the chromogenic dye in the TBX agar is rapidly oxidised to indigo which is insoluble and builds up within the cells resulting in blue-green E. coli colonies. 5.3. Apparatus Filter apparatus and vacumn pump Sterile membrane filters, 47mm cellulose based, 0.45 m nominal pore size. Filter pads Serological pipettes, 10 ml and 1 ml. Smooth tipped forceps. Petri dishes, 90mm and 50 mm diameter Sterilisation tank and boiling water. 5.4. Media Ringers solution, quarter strength. Membrane Lauryl Sulphate Broth add 1.3 ml of sterilised broth to a Petri pad in a sterilised petri dish and allow the broth to soak into the pad. Pour off any surplus broth and cover the petri dish. Nutrient Agar- pour into 90 ml petri dishes and cool to set Oxidase reagent Tetra-methyl p-phenylenediamine hydrochloride. 0.1 g to 10 ml distilled water. TBX agar pour into sterile 50 ml Petri dishes and cool to set. 5.5. Procedure Dilute samples in quarter strength Ringers solution with volumes and dilutions chosen so that the number of colonies on the membrane filter lies between 20 and 80. For each sample, 1 ml is added to 9 ml Ringers solution in a universal bottle and shaken. Serial dilutions to 10-9 are made from the kitchen waste samples; dilutions to 10-6 are made from the other samples. 1 ml of the diluted sample is filtered through the membrane filter using sterile filtration apparatus under vacuum. The membrane filter is rolled onto the petri pad to minimise the likelihood of trapped air bubbles. The filtration procedure is repeated for the 29

other serial dilutions. The petri dishes are then incubated at 30oC for four hours, followed by 37 oC for 48 hours. The procedure is repeated and the petri dishes incubated at 30oC for four hours, followed by 44 oC for 48 hours. 5.6. Reading results After incubation, count all yellow colonies. Those incubated at 37 oC are regarded as presumptive coliforms; those incubated at 44 oC are regarded as presumptive E. coli. 5.7. Confirmation of results To confirm the presence of coliforms, subculture at least ten characteristic colonies onto nutrient agar and incubate 37 oC for 24 hours. Place 2-3 drops of freshly prepared oxidase reagent onto a filter paper contained in a petri dish. Smear some of the growth from the nutrient agar plate onto the treated filter paper. The appearance of a deep blue colour within ten seconds is regarded as a positive result. Count all colonies giving a negative result as coliform bacteria. To confirm the presence of E. coli, transfer a filter from the presumptive E. coli incubation onto TBX agar. Incubate at 44 oC for up to 18 hours. Count all blue-green colonies as E. coli. The number of confirmed coliforms or E. coli are expressed per ml or gram of sample. 6. Microbial analysis Enterococci 6.1. Introduction Enterococci are regarded as secondary indicators of faecal pollution. Identification of species of enterococci present in a sample may help distinguish between human and animal pollution. 6.2. Definitions Presumptive Enterococci reduce triphenyltetrazolium chloride after incubation to insoluble red formazan to produce red, maroon or pink colonies on membrane enterococcus agar. Confirmation is based on the organism being catalase negative and on demonstration of aesculin hydrolysis on bile aesculin agar incubated at 44oC for up to 18 hours. 6.3. Apparatus Filter apparatus and vacuum pump. Sterile membrane filters, 47 mm cellulose based, 0.45 m nominal pore size. Serological pipettes, 10 ml and 1 ml. Smoothed tipped forceps. Petri dishes, 50 mm diameter. Sterilisation tank and boiling water. 6.4. Media KR Streptococcus agar. Bile aesculin agar. 6.5. Procedure Dilute samples in quarter strength Ringers solution with volumes and dilutions chosen so that the number of colonies on the membrane filter lies between 20 and 80. For each 30

sample, 1 ml is added to 9 ml Ringers solution and shaken. Serial dilutions to 10-6 are made. Filter 1 ml of sample through the membrane filter under vacuum. Repeat for each dilution from the sample. Incubated at 37oC for 4 hours followed by 44oC for 48 hours. 6.6. Reading results After incubation, count all red, maroon or pink colonies that are smooth and convex in shape. These are regarded as presumptive Enterococci. 6.7. Confirmation of results Sub-culture a suitable number of red, maroon or pink colonies onto bile aesculin agar and incubate at 44oC for up to 18 hours (hydrolysis should be visible in 2 hours). Enterococci produce discrete colonies surrounded by a brown or black halo from aesculin hydrolysis.

31

Appendix 2: Lithium Tracer Test 1. Lithium Chloride Tracer Testing Lithium tracer testing was carried out to demonstrate the effectiveness of the mixing system of a digester. Lithium tracer testing identifies any short circuiting of feed to the outlet, determines if there are any dead spaces in the digester and whether a significant volume of the digester is occupied by unmixed material. Lithium Chloride is often selected as a tracer because it is non-biodegradable and non-toxic in the anaerobic digestion process. 1. Determination of the Concentration of Lithium Chloride Tracer Addition A 40% solution of Lithium Chloride was obtained from the suppliers. A sample of the stock solution was analysed by Atomic Absorption Spectrophotometry, to accurately measure its concentration. 2. Tracer Test Procedure Lithium chloride solution was added so as to obtain a 2 mg l-1 solution when diluted in the anaerobic digestion tank. Prior to addition of the solution, the inlet and outlet pipes of the digester were cleared with a rodding pipe to ensure rapid flow of the Lithium Chloride dose. Immediately prior to dosing, a 1000 ml sample was taken from the digester outlet pipe. The digestate sample was mixed and a 100 ml aliquot decanted into a 125 ml sample bottle. The remaining 900 ml was returned to the digester. A flexible one-inch pipe (approximately 1 metre in length) was inserted into the digester. The pipe was flushed with 1000 ml of water to ensure that the pipe is not blocked. A stop watch was set to 00.00H and started as the full dose of Lithium Chloride solution was added to the digester. The pipe was immediately flushed with 1000ml of water and then removed. Digestate samples were taken every five minutes for the first two hours, then every ten minutes for the next two hours, and every 15 minutes for the following six hours. Over the next four days, 18 samples were taken between 9.00H and 17.00H, and over the following four weeks, three samples a day were taken (over a five day week). 3. Analysis of Results Each digestate sample was analysed for Lithium (mg l-1) using a standard acid digestion method and Atomic Absorption Spectrophotometry. Three parameters are established as a result of the above sampling and analysis: Mixing Dispersion Time the time taken for the Lithium to be fully dispersed on day one Effective Digester Volume the actual volume into which the Lithium is dispersed (based on the Lithium concentration at the end of day one) Digester Hydraulic Retention Time this is calculated from the decay curve of theLithium from the end of day one to the last day of sampling. It is based on the Effective digester volume, not the nominal digester volume. The actual hydraulic retention time is therefore normally shorter than the nominal hydraulic retention time.

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The hydraulic retention time was calculated from the tracer washout curve: Or Where: C = Lithium Concentration at time t (mg l-1) C0 = Lithium Concentration at time zero, (mg l-1) t = Time, days HRT = Hydraulic Retention Time, days Other parameters were estimated as part of the analysis work: Digester feed rate calculated by dividing the effective digester volume by the hydraulic retention time. Digester Total solids Concentration the total solids of each digester as measured by laboratory analysis. Specific Digester Loading rate the specific digester loading rate for each digester, which is measured as the mass of volatile solids loading per cubic metre of digester capacity per day, was estimated from the digester loading rate, the effective digester volume and the total solids concentration in the digester. In order to arrive at the estimate, it was assumed that the volatile concentration of the raw sludge is 80% and that the percentage volatile solids reduction by the digestion process is 45%. C= C0.e-t/HRT HRT = -t/ln (C/C0)

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Appendix 3: Kitchen Waste Collection 1. Source Separation Scheme

1.1 Selection of participants At the start of the study, collection of approximately 80 bags of kitchen waste from Burford village was already underway. Householders had opted into a previous scheme, which ran from October 1999 to March 2001. Other householders in Burford village and new households in Tenbury Wells were targeted in order to bring additional kitchen waste into the new project. Each household received a promotional recruitment leaflet and an educational leaflet, explaining the need to increase the collection of source-separated kitchen waste and the details of the project. Burford village was targeted on 26th February and 7th March 2002. 100 households in Tenbury Wells were targeted on 16th July and 31st July 2002. In addition, the opportunity to participate in the project was publicised through an advertisement in the local newspaper. 1.2 Provision to the householder Each householder was supplied with one blue 15 litre bucket and lid, a six months supply of white plastic liners, information regarding the collection day and an educational leaflet describing the correct way to separate the waste. An annual update of the scheme and information regarding its success was issued to each participating householder in the form of a newsletter. The newsletter described the collection service to date, the amount of waste diverted from landfill, and again described the separation of kitchen waste to reaffirm the earlier instructions. Weekly collection was provided every Friday morning between 08.00 and 10.00 hours. 2. Kitchen Waste Preparation

On arrival at the digester plant, the kitchen waste bags were counted and each bag weighed using an electronic spring balance. The waste was visually inspected and any rogue material such as metal, plastic or sanitary material was removed. Rogue material and all plastic bags were weighed at the end of the process to calculate the weight of waste produced. The kitchen waste was tipped into the shredder hopper and shredded into a storage container. Any additional rogue material was removed from the storage container at this stage. The received kitchen waste was approximately 23% total solids and 77% water. The waste must be diluted with either digestate or water to allow the feed to be pumped in the digester system. The volume dilution was calculated from the total solids of the kitchen waste and the total solids of the diluting liquid to obtain a feedstock of 12.5% total solids. For example to obtain a feedstock of 12.5% total solids using kitchen waste at 23% total solids, and digestate at 5% total solids, 100 kg of kitchen waste was mixed with 140 kg of digestate obtained from the collection tank. The feed for the mesophilic digester was mixed with digestate from the mesophilic collection tank and the same procedure was followed for the thermophilic digester system. Samples of the kitchen waste and mixed feed were taken for analysis of total and volatile solids at this time.

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Source-separation leaflet supplied to each householder

ORGANISE YOUR ORGANICS!


WHAT ORGANICS TO SOURCE SEPARATE? ORGANIC YES PLEASE Vegetable peelings and trimmings Spoilt vegetables Fruit skins and spoilt fruit Cooked and uncooked meat or fish Bones, fat, egg shells Used teabags, coffee grounds Bread and pastries All cooked food waste

NON-ORGANIC NO THANK YOU Garden Waste Plastic bags and bottles (clingfilm) Glass, metal Newspapers and magazines Dog faeces or cat litter Nappies or sanitary waste Pharmaceuticals and cosmetics

Please could you put out your source separated kitchen waste every FRIDAY MORNING by 8.00 am. If you need any advice or have a query, please phone Christine or Lisa on ***** *******.

Address: ****************************************

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Source separated kitchen waste: 14 month collection period Date Collection week Weight of Collected bag (kg) Total number of bags Average Bag weight (kg) Date Collection week Weight of Collected bag (kg) Total number of bags Average Bag weight (kg) Date Collection week Weight of Collected bag (kg) Total number of bags Average Bag weight (kg) Date Collection week Weight of Collected bag (kg) Total number of bags Average Bag weight (kg) Date Collection week Weight of Collected bag (kg) Total number of bags Average Bag weight (kg) 10.08.01 1 210.4 73 2.88 26.10.01 12 229.1 70 3.27 11.01.02 23 291.6 85 3.43 29.03.02 34 154.3 70 2.20 14.06.02 45 234.3 84 2.79 17.08.01 2 212.3 71 2.99 02.11.01 13 273.5 85 3.22 18.01.02 24 231 82 2.82 05.04.02 35 201 79 2.54 21.06.02 46 230.6 82 2.81 24.08.01 3 194 66 2.94 911.01 14 245.1 80 3.06 25.01.02 25 207 74 2.80 12.04.02 36 266 83 3.20 28.06.02 47 186.7 74 2.52 31.08.01 4 164.6 60 2.74 16.11.01 15 228.8 74 3.09 01.02.02 26 208 76 2.74 19.04.02 37 246.4 86 2.87 05.07.02 48 190.2 76 2.50 07.09.01 5 206.5 81 2.55 23.11.01 16 235.9 71 3.32 08.02.02 27 210 79 2.66 29.04.02 38 229.2 83 2.76 12.07.02 49 186 78 2.38 14.08.01 6 257.4 78 3.30 30.11.01 17 222.2 81 2.74 15.02.02 28 197.8 74 2.67 03.05.02 39 219.2 83 2.64 19.07.02 50 205.6 78 2.64 21.09.01 7 265.7 82 3.24 07.12.01 18 227.8 80 2.85 22.02.02 29 221.3 80 2.77 09.05.02 40 156.6 70 2.24 26.07.02 51 311.6 111 2.81 28.09.01 8 237.5 74 3.21 14.12.01 19 216.9 73 2.97 01.03.02 30 204 78 2.62 17.05.02 41 205.4 72 2.85 02.08.02 52 316.1 116 2.85 05.010.01 9 238.2 76 3.13 21.12.01 20 207.5 76 2.73 08.03.02 31 219.6 79 2.78 24.05.02 42 190.3 80 2.38 09.08.02 53 359.9 121 3.10 12.10.01 10 253.8 82 3.10 28.12.01 21 129.7 35 3.71 15.03.02 32 216.4 82 2.64 31.05.02 43 240.3 85 2.83 16.08.02 54 344.8 114 2.85 19.10.01 11 235.9 76 3.10 04.12.01 22 283.7 71 4.00 22.03.02 33 222.5 80 2.78 07.06.02 44 180.3 70 2.58 23.08.02 55 392.5 110 3.44 Total 2476 819 33.2 Total 2500 796 35.0 Total 2429 869 30.7 Total 2289 861 29.1 Total 2958 1044 30.7

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Source separated kitchen waste: 14 month collection period (continued) Date Collection week Weight of Collected bag (kg) Total number of bags Average Bag weight (kg) Date Collection week Weight of Collected bag (kg) Total number of bags Average Bag weight (kg) Date Collection week Weight of Collected bag (kg) Total number of bags Average Bag weight (kg) Total Kitchen Waste colllected Total number of bags collected Average weight per bag 30.8.02 56 337.5 120 2.81 15.11.02 67 341.6 112 3.05 31.01.03 78 369 119 3.10 06.09.02 57 336 114 2.95 22.11.02 68 360.6 120 3.01 07.02.03 79 359 123 2.92 23,182 7852 2.95 13.09.02 58 359.1 120 2.99 29.11.02 69 339.4 118 2.88 14.02.03 80 366 118 3.10 kg kg 20.09.02 59 441.8 122 3.62 06.12.02 70 365 121 3.02 21.02.03 81 352 114 3.09 27.09.02 60 363.3 117 3.11 13.12.02 71 333.5 117 2.85 28.02.03 82 348 122 2.85 04.10.02 61 351.5 108 3.25 20.12.02 72 344.6 124 2.78 07.03.03 83 365 121 3.02 11.10.02 62 385.2 127 3.03 27.12.02 73 0 0 0.00 14.03.03 84 387 125 3.10 18.10.02 63 357 121 2.95 03.01.03 74 492 141 3.49 21.03.03 85 348 119 2.92 25.10.02 64 366.4 119 3.08 10.01.03 75 352.7 117 3.01 01.11.02 65 343.7 113 3.04 17.01.03 76 373.8 115 3.25 08.11.02 66 328.9 118 2.79 24.01.03 77 361.6 118 3.06 Total 3970 1299 33.6 Total 3665 1203 30.4 Total 2894 961 24.1

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Appendix 4: Hygiene Regime Hygiene Regime It was vital to set up a hygiene regime in order to ensure that there was no cross contamination of pathogens between the treated and untreated waste. 1. For each digester stream, clean and dirty areas were designated Clean area contains treated digestate and no contact with untreated kitchen waste was permitted Dirty area contains untreated kitchen waste and digestate that has had less than six hours treatment in the digester. 2. Labelled tools were assigned to each area and digester stream.

1.1. Collection tank cleaning The digestate collection tank was emptied using a submersible tank. When as much digestate as possible had been removed, the tank was filled with water and mixed to aid removal of remaining material. The diluted material was pumped out of the tank and this procedure was repeated. The tank was then washed with a pressure hose and pumped out. The inner surface of the tank was scrubbed with dilute disinfectant, then pressure washed again and pumped out. A dilute solution of sodium hydroxide was used to aid removal of organic material. After soaking for five minutes, the tank was rinsed with water and pumped out. Following this, it could again be filled with digestate.

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