**4. Elimination of potential pathogenic bacteria in municipal sludge**

#### **4.1. Sampling: experimental procedure**

Sample collection, transport, and storage are crucial when studying the effect of electron beam irradiation on microbial population found in municipal sewage sludge. In these experiments, sewage sludge samples were collected in two separate batches. First batch contains pretreated municipal sewage sludge or influent samples, and a second batch is made of municipal sewage sludge treated with electron beam irradiation or effluent samples. In the case reported here, since the sludge was treated with different doses of electron beam irradiation, samples were collected prior to (influent) and after (effluent) irradiation of sludge at each dose. Several 100 ml influent and effluent samples of sewage sludge were harvested in sterile-caped plastic vials for bacterial count and survival. Each sample was then placed on ice immediately after collection and transported in an isotherm ice container (a cooler) from the electron beam irradiation facility to the microbiology laboratory for microbial analysis. For accurate obser‐ vation of the direct effect of electron beam irradiation on bacterial population, samples should be analyzed as soon as possible after treatment.

### **4.2. Sample analysis using membrane filtration method: experimental procedure**

Each sample was thoroughly mixed, and serial dilutions were performed in 1× phosphatebuffered saline. Influent samples were diluted up to 10−8, while effluent samples were diluted up to 10−6. Diluted samples were filtered using disposable filter funnels. For filtration, 10 ml of the diluted sample was transferred with a sterile pipette into the middle of a sterile 45 mm (diameter) and 0.45 μm (pore size) gridded membrane filter. After filtration, the filter was washed with three volumes of 1× phosphate-buffered saline. The filter was then removed and transferred on a 50-mm (diameter)-padded Petri dish plate containing 2 ml of culture medium for total heterotrophic bacterial (THB), total coliform (TC), and fecal coliform (FC) counts. In order to perform THB counts, mHPC Heterotrophic medium was used and for TC counts, mEndo medium was used, while m-FC medium supplemented with Rosolic acid was used for FC counts. Plates were placed in plastic bags containing moistened paper towels and trans‐ ferred in an incubator. Heterotrophic plates were placed in an incubator for 48 h at 35 ± 0.5°C, and TC plates were incubated for 22–14 h at 35 ± 0.5°C, while FC were incubated at 44.5 ± 0.2°C. Known positive and negative controls were used in order to verify accuracy of analytical procedures for identification and counts of heterotrophic, TCs, and FCs. Thus, for TC media, *Escherichia coli* and *Enterobacter aerogenes* were used as positive controls, while *Staphylococcus aureus* and *Pseudomonas aeruginosa* were used as negative controls. The positive control for the FC media was *E*. *coli*, and the negative control was *E*. *aerogenes*. Prior to testing, test organisms were grown in tryptone soy broth and incubated overnight at 37°C. After growth, these cultures were treated according to the procedure used for sewage sludge samples. Only dilution plates with a density of 20–100 colonies were counted. FC colonies appeared as different shades of blue, while other non-FC colonies appeared as gray or cream-colored. Bacterial counts were performed in triplicate to verify the reproducibility of results for total heterotrophic counts, TC counts, and FC counts.

#### **4.3. Results and discussion**

currents to compensate for this effect. The dose was determined then from temperature increase of the sludge by measuring the temperatures at the input and exit ports of the irradiation setup. **Table 2** presents results of the temperature increments and dose measure‐ ments as a function of the beam currents for the sludge sample running through the delivery

**Beam current (mA) Temperature increase (°c) Dose (kGy)**

**Table 2.** Irradiation conditions used to achieve targeted doses. Sludge samples were flowing at a rate of 30 gpm [7].

Sample collection, transport, and storage are crucial when studying the effect of electron beam irradiation on microbial population found in municipal sewage sludge. In these experiments, sewage sludge samples were collected in two separate batches. First batch contains pretreated municipal sewage sludge or influent samples, and a second batch is made of municipal sewage sludge treated with electron beam irradiation or effluent samples. In the case reported here, since the sludge was treated with different doses of electron beam irradiation, samples were collected prior to (influent) and after (effluent) irradiation of sludge at each dose. Several 100 ml influent and effluent samples of sewage sludge were harvested in sterile-caped plastic vials for bacterial count and survival. Each sample was then placed on ice immediately after collection and transported in an isotherm ice container (a cooler) from the electron beam irradiation facility to the microbiology laboratory for microbial analysis. For accurate obser‐ vation of the direct effect of electron beam irradiation on bacterial population, samples should

**4. Elimination of potential pathogenic bacteria in municipal sludge**

**4.2. Sample analysis using membrane filtration method: experimental procedure**

Each sample was thoroughly mixed, and serial dilutions were performed in 1× phosphatebuffered saline. Influent samples were diluted up to 10−8, while effluent samples were diluted up to 10−6. Diluted samples were filtered using disposable filter funnels. For filtration, 10 ml of the diluted sample was transferred with a sterile pipette into the middle of a sterile 45 mm (diameter) and 0.45 μm (pore size) gridded membrane filter. After filtration, the filter was washed with three volumes of 1× phosphate-buffered saline. The filter was then removed and transferred on a 50-mm (diameter)-padded Petri dish plate containing 2 ml of culture medium

**4.1. Sampling: experimental procedure**

be analyzed as soon as possible after treatment.

2.3 0.6 2.7 5.8 1.6 6.7 11.5 3.1 13.2 23.0 6.1 25.7 27.6 7.3 30.7

system.

238 Radiation Effects in Materials

Bacterial counts before and after irradiation were performed with the electron beam at doses 2.7, 6.7, 13.2, 25.7, and 30.7 kGy. The counts were done specifically for THB, TC, and FC. **Figure 5** shows the effect of electron beam irradiation on bacterial survival in municipal sewage sludge after treatment. It appears that THB, TC, and FC counts decreased in a dose-dependent manner. This decrease in bacterial population is directly associated with the ionizing effect of electron beam irradiation that damages bacterial DNA and biomembranes, and the production of reactive oxygen species which also damage cell components. A similar observation was recently made by Cao and Wang [8] when they treated municipal sludge with electron beam irradiation. However, these authors did not count specific types of bacteria.

**Figure 5.** Effect of electron beam irradiation on bacterial survival in municipal sewage sludge samples. (a) Survival of total heterotrophic bacteria, (b) survival of total coliforms, and (c) survival of fecal coliforms [7].

Looking more into details, it was shown that when irradiating sludge with electron beam, a dose of 2.7 kGy, 93.3 ± 8.5% THB survived the treatment, while only 21.1 ± 11.4% of TC and 67.2 ± 1.8% of FC survived at the same irradiation dose. At a dose of 6.7 kGy, while 31 ± 15% of THB survived the treatment, only 0.85 ± 0.23% and 1.85 ± 0.65% of the initial populations of TC and FC survived, respectively. At doses of 13.2 kGy and above, neither TC bacteria nor FC were detected. Nevertheless, at a 13.2 kGy irradiation dose, 8.9 ± 1.3% of THB from the initial population survived the treatment. At a dose of 25.7 kGy and above, no significant THB from the initial population were left in treated sewage sludge samples [7]. **Table 3** summarizes bacterial counts per gram of sludge dry weight at different electron beam doses. From these results, D10-values were determined as 8.94, 3.16, and 3.17 kGy for THB, TC, and FC respec‐ tively. D10-values are defined as doses necessary to kill 90% of the bacterial populations in the sample for irradiation conditions applied, or the dose needed to reduce the bacterial popula‐ tion by a factor of 10. A close look at **Table 2** shows that dose 6.7 kGy reduces the FC counts to 180 colony forming unit (CFU) per gram of sludge dry weight, a count that is within the Environmental Protection Agency (EPA) norm to classify such treated municipal sewage sludge as class A sludge utilizable for land application in agriculture [9]. However, from the D10-value determined for FC, based on initial population of FC in influent samples, the dose required to convert this sludge to class A was estimated to be 4.5 kGy. Although no previous work similar to this one is known to perform a comparison with our estimated D10-value, nevertheless, water-based and surface membrane *Bacillus* spore killing D10-values were reported to be 1.3 and 1.53 kGy, respectively [10, 11]. These values are about twice lower than the 3.17 kGy determined in our case. This difference could be attributed to the presence of a large amount of organic and inorganic materials that make our sample relatively thick and slightly viscous compared to water and a surface membrane.


**Table 3.** Bacterial counts in sludge samples at different irradiation doses [7].
