Study of helminth eggs (Ascaris suum) inactivation by anaerobic digestion and electrochemical treatment

Anaerobic digestion: biochemical methane potential (BMP) assay

Experimental setup. The biochemical methane potential (BMP) assay was performed in serum bottles (130 mL) to co-digest faecal sludge (Obtained after solid-liquid separation of septic tank sewage from the sewage treatment plant at Baina, Goa) and food waste (Collected from one of the messes of BITS Goa). The ratio of food waste to sludge in the bottles was 5:1. Before seeding the serum container, the sludge's total solid (TS) and volatile solid (VS) contents were evaluated. Each bottle was subjected to a loading rate of biomass of 1.5kg VS/m³; the amount of faecal sludge and food waste to be added to the bottles to achieve desired loading rate was calculated using the VS values. VS was first calculated in kg/m³ for a 5:1 ratio and then converted to grams/100mL as the final bottle volume was 100mL. Macro and micronutrients, sodium bicarbonate, digested from a large-scale anaerobic reactor (Plug flow reactor of 60 m³ capacity constructed at BITS Goa for the treatment of food waste to produce biogas) as inoculum were added, and the final volume was raised to 100 mL by adding distilled water. A. suum eggs were procured from Excelsior Sentinel, USA, a mixture of viable and non-viable eggs (Figure 1). Eggs of concentration 8 eggs/mL with 63% viability were spiked in the BMP bottles. The experiment was performed using two bottles in each set; one bottle from each set was taken out at each sampling point (Day 0, 3, 6, 9, 15, 20, 25, 30, 35, 40, and 45) for analysis, and another bottle along with the remaining bottles from the set was used to take the gas reading every day. When the bottle was taken out at each sampling point, a gas reading from one bottle was considered a duplicate reading to perform analysis. The setup was maintained at room temperature (28 ± 2°C).

Figure 1.

(a) and (b): The viable and non-viable mixture of procured Ascaris suum eggs.

Parameter analysis. The different experimental parameters analyzed are given in Table 1. Biogas production was estimated daily by measuring the gas volume generated using a water displacement unit. A water displacement unit was made in the laboratory: a 5000 mL glass beaker was taken, and a 100 mL measuring cylinder was attached inside in an inverted position. The beaker was filled with distilled water until the zero mL mark on the cylinder, and gas from the bottle was passed inside the cylinder using a syringe with an IV tubing set. As the gas passed into the cylinder, it displaced the water, and the reading was recorded in mL. pH, TS, and VS analyses were conducted at each sampling point. PH was checked using an Oakton pH meter. The pH probe was dipped into the sample and the reading was taken. Readings were taken in duplicate. To plot a graph, standard deviation was calculated using the duplicate pH reading. For TS and VS analysis, the weight of the empty crucible was recorded, then the sample was added and the total weight was recorded. Sample weight was calculated by subtracting the crucible weight from the total weight (For the solid sample around 5 g of sample and for the liquid sample around 10 mL of sample was taken). Crucibles containing samples were kept at 105°C for 12 hours and the weight was recorded. Then the crucibles were ignited in a muffle furnace at 550°C for four hours and weighed once they had cooled down. TS was calculated by subtracting the crucible weight after the 105°C readings. VS was calculated by subtracting the 550°C weight values from the 105°C weights. Both TS and VS was then converted to per gram and then to percentage. To plot a graph, standard deviation was calculated using percentage values of both TS and VS.

Biogas composition (CH₄, CO₂, and H₂S) was analyzed using gas chromatography (GC). The GC instrument (Trace 1110, Thermofisher Scientific) was equipped with a packed stainless-steel column of spherocarb support, a thermal conductivity detector (TCD), and hydrogen as the carrier gas. The injector temperature was set to 150°C and the detector temperature to 185°C. Gas sample was taken from BMP serum bottles using a 2 mL syringe and injected into the GC instrument. Gas composition was determined by following the steps in the software (Chrom-Card data system, Version 2.12, August 2014) present in the instrument itself.

Volatile fatty acid (VFA) analysis was conducted using the titration method. A 5 mL sample was taken from the opened serum bottle, and the volume was raised to 100 mL using distilled water. The diluted sample was titrated in a glass beaker using 0.1 N HCl until the pH reached 3, and the burette reading (A) was recorded. Then 0.1 N NaOH was added until the pH reached 6.5, and the burette reading (B) was recorded. The following formula was used to calculate VFA (mg/L)

For helminth eggs analysis, the sample was taken in a plastic beaker and ammonium bicarbonate solution was added; the sample was mixed on a magnetic stirrer (Cole-Parmer- Stuart-UC152D) for 20 min. The resultant solution was passed through a 100-micron sieve (Analysensieb test sieve, 200 mm diameter, Fritsch, Germany) kept on top of a 20-micron sieve. The sample was washed with tap water, and the contents of the 20-micron sieve were placed in four 15 mL centrifuge tubes and centrifugated at 3000 rpm in a swing out rotor centrifuge (Eltek, MP 800) for 10 min. After centrifugation, the supernatant was discarded and zinc sulphate solution of 1.3 specific gravity was added to the pellet to the 14 mL mark with 3 ml solution added at a time and mixed on a vortex (Neuation, Digital Vortex Mixer iSwix VT) and centrifuged again at 2000 rpm for 10 min. The supernatant was carefully placed in a small 20-micron sieve (100 mm diameter) and washed thoroughly. The contents of the sieve were transferred to a centrifuge tube, and centrifugated at 3000 rpm for 10 min. The pellet was made into suspension by adding distilled water (1–2 mL) for microscopy using distilled water (UKZN PRG helminth method (Pebsworth et al., 2012). Using the ZEISS Primo Star microscope, eggs were observed and counted under 10X and 40X magnification. Images were captured by using iDS uEye Cockpit software (Version 4.96.1) which comes with the microscope. All parameters were examined in duplicate every third day for the first 15 days, then every fifth day until day 45. All the methods in detail are uploaded on Dryad (Mutnuri & Patil, 2023).

Statistical analysis. All the data were checked for normality and homogeneity of variance. The relationship between A. suum inactivation and all the other parameters was studied using correlation analysis. Statistical tests were performed using SPSS (IMB SPSS Statistics 25) software.

Electrochemical treatment

Experimental setup and experiment. The laboratory-scale electrochemical cell (EC) was set up in a 1000 mL beaker (Figure 2). A titanium plate was used as an anode electrode, coated with 6 µm thick mixed metal oxide (Ruthenium, Iridium, and Titanium in 70%, 20%, and 10%, respectively). The cathode electrode was made of SS 304 stainless-steel mesh. A variable DC supply unit (Laboratory DC power supply, Gwinstek, GPS-4303) was used to apply a consistent voltage. The electrochemical cell solution was continuously mixed with a magnetic stirrer (SPINIT Motorless Magnetic Stirrer, Tarsons) to generate hypochlorite, which was then employed as a disinfectant for inactivating A. suum eggs.

Figure 2. Electrochemical cell set up at lab scale.

Hypochlorite generation using NaCl has been done with different electrochemical processes (Asokan & Subramanian, 2009; Gogoi et al., 2022; Spasojevic et al., 2015). This study generated hypochlorite as described in Gogoi et al., 2022 with a few modifications. Electrolysis of 2% NaCl solution was performed for two hours, producing 4700 ppm hypochlorite. The generated hypochlorite was diluted in various ratios with distilled water (1:5, 1:10, 1:20, 1:30, and hypochlorite without dilution). Helminth eggs (8 eggs/mL; 86% viability) were spiked into all diluted solutions, and helminth inactivation was observed under a microscope at 15 min, 30 min, hourly intervals between one and six hours, and 24 hours.

Inactivation of A. suum eggs by anaerobic digestion

Figure 3 (a) depicts the biogas produced in the BMP assay. On the first day, the gas produced was between 112 and 122 mL, and on day 30, the overall gas generation (cumulative) was 1218 to 1389 mL. After 25 days, gas production began to decline; thus, recording readings were discontinued at 30 days. The produced gas was then examined using GC.

Figure 3.

a) Cumulative biogas production, variations in the parameters b) pH, c) TS, d) VS, e) VFA, f) CH₄, CO₂, g) H₂S, h) A. suum inactivation eggs/mL.

Throughout the experiment, the pH of the biomass remained approximately neutral (7–8) (Figure 3(b)). It was 7.83 at the start of the experiment (day 0), but it dropped to 6.98 at the next sampling point (day 3). It then went up to 7.37 and remained between 7.3 and 7.5 for the remainder of the incubation period.

The amount of TS and VS in the biomass reduced as the incubation progressed (Figure 3(c) and (d)). On day 0, TS was 3.40%; by day 45, it had dropped to 2.18%. VS followed a similar pattern, peaking at 63.53% on day 0 and dropping to 50.33% on the 45th day of incubation.

The VFA production in the biomass was highest on day 3 (9181 mg/L), which then decreased as the incubation period proceeded (Figure 3 (e)), dropping down to 2572 mg/L, observed on the last sampling day.

The trends for CH₄ and CO₂ were diametrically opposed (Figure 3 (f)). CH₄ climbed to 69% until the 20th day, then declined to 54.33% until stabilizing on the 40th day. CO₂ levels were high at 72.38% at the first sampling point (day 3). It declined (28.38%) until the 20th day of incubation and then increased slightly until the experiment ended. Throughout the trial, H₂S displayed an uneven trend (Figure 3 (g)). The maximum H₂S concentration (1.63%) was found on day 9, while the lowest (0.61%) was observed on day 35.

The total number of viable A. suum eggs dropped over the course of the experiment, contributing to the increase in non-viable eggs (Figure 3 (h)). The initial viable egg count was roughly 4 eggs/mL, reducing to 0.15 eggs/mL on the 35^th day (which meets the discharge standard according to WHO, 2006). Inactivation of eggs was relatively low for the first 15 days (2.35 eggs/mL) and then continued to increase, with 98% inactivation achieved by the 35th day of incubation. There was not much additional variation in the number of viable eggs after day 35 (0.14 eggs on the 40^th day); inactivation remained at 98%, so the experiment was stopped at 45 days. After 35 days, the number of non-viable eggs dropped marginally; this could be attributable to the complete disintegration of a few dead eggs that were not counted. Figures 4 (a–f) show representative pictures of A. suum on day 45.

Figure 4.

A. suum viable eggs (a), (b) and non-viable eggs (c–f) on 45^th day of incubation.

Effects of test parameters on A. suum eggs inactivation

Since the data for a few testing parameters was not normally distributed, Spearman's correlation analysis was used to individually analyze the relationship between the number of non-viable eggs and each test parameter. The analysis results demonstrated a positive association between pH and non-viable eggs (rs = 0.465, p = 0.022), indicating that the number of non-viable cells will also increase as the pH rises. TS and VS had a substantial negative connection with non-viable eggs (rs = -0.752, -0.607, and p = 0.000, 0.002, respectively). VFA also had a strong negative correlation with non-viable eggs (rs = -0.840, p = 0.000), indicating that increasing VFA concentration alone will not contribute to the inactivation of helminth eggs. The CH₄ and CO₂ values were not statistically significant (p = 0.474, 0.563, respectively), indicating that the effect is insignificant in this case. H₂S was negatively correlated with non-viable eggs (r = -0.555, p = 0.011).

A. suum inactivation by the electrochemical process

None of the different hypochlorite concentrations demonstrated evidence of helminth eggs being inactivated until six hours into incubation in the diluted hypochlorite solution. Only 10% inactivation was achieved in the 1:5 ratio (940 ppm) solution after a 24-hour incubation period. When exposed to absolute hypochlorite (without dilution), some of the A. suum eggs were quickly inactivated and over 24 hours, their inactivation was maximized. Table 2 shows the parameter values, and Figure 5 shows A. suum egg images before and after inactivation in hypochlorite solution.

Figure 5.

A. suum viable eggs (a–c), and non-viable eggs (d–f), from the EC experiment. (g, h and i), show the dissolution of the outer part of the eggshell and subsequent membrane rupture, resulting in complete inactivation of the egg in hypochlorite solution.