MATERIALS AND METHODS

3.2.1 Survey sites and study population

The peri-urban district of location x was used for the collection of pit latrine (n=11) and unsewered public toilet (n=3) samples in February/March 2013. Pit latrines in this region were raised above ground level and had no vent pipes present. Community ablution blocks consisted of 4-12 flush toilets, urinals as well as running water used for washing and showering. This waste was then stored in lined holding tanks that were set below ground level.

A pilot scheme (Clean Team, 2012) using specially designed portable toilets and a regular collection service was used as a site for portable toilet sludge characterisation in Kumasi, Ghana in July/August 2013. Portable toilets (n=36) were designed to be urine diverting toilets with only the faeces fraction collected, in a detachable container at the base of the toilet structure, and collections took place at least once every 4 days from households (average household users 4.2). Faeces were collected in this container which contained a commercially available toilet chemical additive (Active ingredients: Pentane 1,5 diol 5-10%, 2-Bromo-2-Nitropropane-1,3-diol <5%) which was diluted in 5 -7 L of water in order to prevent odour issues. Collected portable toilet waste was subsequently emptied into an IBC (Intermediate Bulk Container, 1 m3 volume) (n=1) where it was stored for approximately 1 month prior to final treatment and disposal.

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Institutional ethical approval (Cranfield University 109-2013) was obtained for the sampling and analysis of faecal sludge in both case study locations: location x and Kumasi, Ghana.

3.2.2 Analytical methods

On-site measurements of pH and temperature were recorded using a portable YSI G3 60 meter (YSI Ltd., Hampshire, UK) and the oxidation reduction potential (ORP) was measured using an ORP15 pen (model number I662-0121, VWR International, Leuven, Belgium). Bulk FS samples were taken (containing a combined mixture of municipal solid waste and faecal material) and aliquots of 2 L were put into sealed plastic bags and stored on ice in a cool bag before being transported to the laboratory (approximate journey time of 1 hour) where they were refrigerated (4oC ± 0.5°C) on arrival. A plastic sample bag was also filled with FS and was immediately frozen (-20°C ± 0.5°C) once at the laboratory.

In the laboratory set up at each case study location 2 L of each bulk sample was homogenised using a mechanical blender according to the methods of Buckley et al. (2008), any material that could not be blended was discarded and classified as municipal solid waste (MSW). All samples were analysed in triplicate and the mean and standard deviation recorded. Total solids (TS), volatile solids (VS), total nitrogen (TN), ammonium (NH4-N) and faecal coliforms

were all determined on the homogenised wet sample. In addition a frozen aliquot of the complete sludge sample was stored and used for biochemical methane potential (BMP). The TS and VS were measured according to standard methods (APHA, 2005), TN was determined after digestion according to the Koroleff method (analogous to EN ISO 11905-1) and was measured photo-metrically (analogous to DIN 38405 D9) and NH4-N was measured

according to DIN 38406 E5.

The homogenised sludge sample was also centrifuged for 10 minutes at 5000 g (Hettich Zentrifugen, Tuttlingen, Germany) after which the supernatant was filtered through a 0.45µm filter (Sartorious, Epsom, UK) in order to give the soluble solid free fraction. Analysis was undertaken for CODsol, nitrate (NO3-N),

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total phosphorus and potassium according to standard methods (APHA, 2005). An aliquot of 5 mL was immediately frozen (-20ºC) awaiting analysis for soluble magnesium and volatile fatty acid concentrations.

A range of six volatile fatty acids (VFA) concentrations were measured and determined on the soluble solid free fraction using high pressure liquid chromatography (HPLC) (535 Kontron, Bio-TEK, UK) with a Bio-Rad fermentation column (Cat 125-0115) 300 x 7.8 mm maintained at 65ºC, with a UV detector at 210 nm. The sample volume was 50µl. All samples for VFA analysis were first acidified with concentrated H2SO4 to pH <2 according to

Parawira et al. (2004) and a hydraulic flow rate of 0.8 mL/min used. An external multi-level calibration range from 0.1 g/L to 5 g L was used to quantify acetic, propionic, n-butyric, i-valeric and n-valeric acids.

The solid fraction was obtained by drying c.0.5 L of FS at 40oC±0.5 for 48 hours, to avoid potential volatilisation of nutrients and prevent further degradation of the samples. The samples were then sealed in plastic bags to await subsequent analysis. Quantitative elementary analysis was undertaken using a Vario EL (Elementar, Hanau, Germany) for % C, H and N according to ISO 10694. Total P in the dried solids fraction was measured according to US EPA Method 3051 and determined photometrically (Helios Gamma, New Brunswick, USA). Potassium (K), magnesium (Mg) and calcium (Ca) as well as heavy metals were determined after microwave digestion with a nitric/hydrochloric acid mixture and was measured using Atomic Adsorption Spectroscopy (AAnalyst 800, Perkin Elmer, Waltham, USA) according to APHA (2005).

Biochemical methane potential (BMP) assays were carried out according to Owens and Chynoweth (1993) and Angelidaki at al. (2009). The anaerobic inoculum was collected from a mesophilic digester at a sewage treatment works (population equivalent (PE) of 288,000) and a stock solution of micronutrients (according to Gonzalez-Gil et al. (2001)) was added to each assay. Each assay was flushed with N2/CO2 (80:20% as volume) after transfer of inoculum and

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controlled shaker (37.5oC ± 0.5: 150 rev min-1)( (Excella E24, New Brunswick Scientific, Edison, USA). Cellulose was used as a positive control in all experiments and assays were carried out in quadruplicates. Blanks were run with inoculum only with no substrate addition and biogas production from the inoculum was subtracted from methane production values of tested substrates. Total gas volumes were calculated by water displacement. The production of biogas was given at standard temperature and pressure according to Angelidaki et al. (2009). The % methane (CH4) in biogas samples was measured using a

Servomex 1440 (Zoetermere, Netherlands) gas analyser.

Potential methane inhibition caused by the chemical toilet additive in use was investigated by determining the IC50 values of the two main active ingredients of the chemical additive (Glutaraldehyde (Pentane 1,5 Dial) and Bronopol (2- Brono-2-Nitropropane-1,3 Dial). In order to test the inhibitory effect of these chemicals a primary sludge from the 288,000 PE sewage treatment plant was used as a substrate and a range of inhibitory and sub-inhibitory concentrations of Glutaraldehyde (10, 50, 100, 500, 5000 and 10000 mg L-1) and Bronopol (10, 50, 100, 500, 1000, 10000 mg L-1) were added to the BMP assays completed as outlined above. Any inhibitory effect was determined by assessing the IC50

value and was compared to a control group with no chemical addition.

Thermotolerant coliform determination, DNA extraction and sequencing were carried out on pit latrine and portable toilet sludge samples only. Thermotolerant coliforms were determined within 4 hours of sample collection using membrane filtration and incubated on membrane lauryl sulphate broth at 44°C ±0.5°C for 14 hours following the standard method for detection and enumeration of coliforms (APHA, 2005). DNA extraction and sequencing was conducted on homogenous sludge samples taken from 11 pit latrines and 3 public toilets as well as additional depth samples from the top (0.05 m), middle (1 m) and bottom (2.5 m) (total pit latrine depth 2.9 m) from one of the pit latrines (S863). One pit latrine was a dry urine diversion pit latrine and was identified as an ECOSAN latrine. All samples were stored at -20°C ± 0.5°C prior to DNA extraction. Methods of DNA extraction, next generation sequencing library construction and PCR amplification are provided in detail in Appendix B. The pooled multiplexed

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library was sequenced using the Illumina Miseq (San Diego, USA) bench-top sequencer at the Centre for Genomic Research, Liverpool.