Analytical procedures

Total solids and volatile solids

Total solids (TS) and volatile solids (VS) analysis were conducted according to the gravimetric method (SM-2540D and SM-2540E), as outlined in Standard Methods for the Examination of Water and Wastewater (APHA, 1995a). TS was analysed by drying 10 g sample weight in an oven at 105 °C for 24 hours, after which they were cooled and weighed. For VS determination, the samples were further dried in a muffle furnace at 550°C for 2 hours. pH measuring was done using an analogue pH/ORP meter (model HI8314-S/N 08586318) calibrated with buffer solution at pH 4 and pH 7.

VFA measurements

Volatile fatty acids (VFA) in wastewater samples can be measured using gas chromatographical methods for organic acids or by titrimetric method (Jobling Purser et al., 2014; Lutzhoft et al., 2014). In this study, the titrimetric method as described in analytical methods for waste water characterisation and evaluation of reactor performance during anaerobic treatment (AGROIWATECH, DeliverableD2) was adopted. The sample, placed in centrifuge tubes was centrifuged for 5 minutes at 5,000 revolutions per minute (rpm). 50 ml of the supemant was put in a beaker. The pH of the solution was adjusted to 6.5 using either 0.1M HC1 or NaOH solution, and thereafter it was titrated with 0.1M HC1 to pH 3. This volume of acid was recorded. The samples were transferred to a digestion flask with glass beads and connected to a condenser. Flask was heated until liquid began to boil and then allowed for three minutes boiling. The heater was then switched off and two minutes were allowed for cooling. The sample was then titrated immediately to pH 6.5 and volume of base recorded. The total VFA values in meq/ 1 added were then calculated using the formula (AGROIWATECH, DeliverableD2):

Where В is the volume of 0.1 M sodium hydroxide required to titrate from pH 3-6.5; C the total volume of titrated sample (ml), 0.1-meq conversion factor, 1000- ml to 1 conversion.

In addition, background acidity correction was carried out to correct for proton acceptors present in the waste water that are not volatile, e.g. humic acids. For this, the prescribed procedure was carried out on fresh UDDT-FS and OMW samples and obtained VFA values subtracted from incubated sample values. The procedure also accounted for actual VFAs in the sample before treatment, so as to set a baseline for VFA build-up after treatment.

From the Total Volatile Fatty Acid (TVFA) concentration, the fraction of ND-VFA was calculated. VFAs are commonly considered to constitute a single weak-acid system with equilibrium constant Ka because of the similarity of their pK values (Lahav & Morgan, 2004; Moosbrugger R. E. etal., 1993). Therefore:

Where: AT = total VFA species concentration (rng/L), HA represents the acidic, protonated species and A^- the ionised form of each acid.

E. coli enumeration

E. coli enumeration was done using the chromocult coliform agar (CCA) technique, which was proved applicable for use in temperate regions (Buckley et ah, 2008; Byamukama et ol, 2000; Frampton et oh, 1988; Manafi & Kneifel, 1989). The CCA (Chromocult; Merck, Darmstadt, Germany) was prepared following manufacturer’s instructions. Homogenised samples were serially diluted (10¹ to 10'⁶) with the peptone buffered water. For each sample dilution, 0.1 ml was spread on prepared chromocult agar plates in duplicate. The prepared plates were then incubated for 24 hours at a temperature of 36±1 °C, after which colony counting was facilitated by use of a colony counter (IUL magnifying glass colony counter, IUL, S.A., Barcelona, Spain). The criteria used for identification were able to identify dark blue- to violet- coloured colonies as E. coli (Byamukama et ah, 2000; Fimiey et ah, 2003). The average numbers of colonies were used to calculate the E. coli concentrations in the samples, expressed in CFU/ g 100 ml of the test sample. In the method, the lowest detection limit is 1000 CFU/L.

The first order reaction coefficients for E. coli removal were calculated using the Chick- Watson model that expresses the rate of inactivation of micro-organisms by a first order reaction.

Where:

C_t = Number of micro — organisms at time t C₀ = Number of micro — organisms at time 0 к = decay constant t = time

A. lumbricoides egg recovery

A. lumbricoides egg recovery was performed according to method developed by Moodley et al.., (2008) and modified by Pebsworth et al.., (2012). Ammonium bicarbonate solution was added to 80 ml of sample in order to wash and dissociate the eggs attached on the particles. It was then passed through a 100 pm sieve onto a 20 pm sieve. Sieve contents were well washed and all material held on 100-pm discarded. Material held on the 20 pm sieve was washed and collected onto autoclaved 15 ml centrifuge tubes, and centrifuged using a bench top centrifuge (EBA 20, Andreas Hettich GmbH &CO. KG, Germany) at 3000 rpm for 5 minutes. Supernatant was discarded and remaining pellets re-suspended in Z11SO4 (specific gravity 1.3), while vortexmg until the 14 ml level. The samples were then centrifuged again at 2000 rpm for 5 minutes. Supernatant was then poured through a small 20-pm filter, and washed off into an autoclaved plastic test tube. It was then centrifuged again at 3000 rpm for 5 minutes. The supernatant was discarded and the egg pellets were transferred into a 50 mL Falcon tube, with de-ionised water added to the 45 ml mark. The Falcon tube was covered with parafilm that was pricked (to allow air exchange within the sample) and then incubated at 28±1 °C for 28 days. Regular checks with de-ionised water additions were conducted in order to account for water lost through evaporation.

After the 28 days incubation, the samples were divided into 15 ml centrifuge tubes and centrifuged for 5 min. at 3000 rpm. The supernatant was discarded and the remaining pellet containing eggs was well mixed using a pipette. 1 ml of sample was transferred on a Sedgewick- rafter counting cell (from Wildlife Supply Company¹¹). The slide was observed under the microscope (AmScope, California, USA) at a magnification of 10 and 40.

Eggs developed to the larval stage, with motile larvae, were considered viable, while all eggs that stopped under any other developmental stage and eggs that presented some kind of deterioration with no motile larvae inside were considered non-viable.

Data analysis

Data analysis was done using Microsoft Excel software. Data obtained from each batch was first analysed by computing the averages of the three trials conducted per batch. Average values of the three batches were then combined by computing their average values, standard deviations and standard errors. The average values of the three batches were then presented in either Table of Figure form.