MICROFLORA INHABITING RAW SEWAGE, SECONDARY EFFLUENT AND DEWATERED SLUDGE IN IBB, YEMEN REPUBLIC الأحياء المجهرية التي تعيش في مياه المجاري والمخلفات الثانوية السائلة والوحل الجاف في محطة التنقية في إب- الجمهورية اليمنية

MICROFLORA INHABITING RAW SEWAGE, SECONDARY EFFLUENT AND DEWATERED SLUDGE IN IBB,

 YEMEN REPUBLIC

Al-Zubeiry, A. H. S.

Microbiology Department, Faculty of Science, Taiz University, Yemen

INTRODUCTION:

Wastewater generated from urban and rural areas after domestic use is a large source of water. It is mainly comprised of water (99,9%) together with relatively small concentrations of suspended and dissolved organic and inorganic solids (Mara and Cairncross, 1989 and UN Department of technical cooperation for development, 1985).

Among the organic substances present in sewage are carbohydrates, lignin, fats, soaps, synthetic detergents, proteins and their decomposition products, as well as various natural and synthetic organic chemicals from process industries.

Sewage sludge (wastewater) is an important source of organic matter (Stranchan., et al.,1983) and plant nutrients. Halderson and Zonz, (1978); Nell et al., (1983) found that the application of sewage sludge increased the nutrient status of the soil. It may increase agricultural production. Sewage sludge also contains pathogenic macro and microorganisms, which can give rise to potential hazard (Abderrahman and Shahlam, 1991) to the health of humans, animals and plants. The health risk associated with wastewater is a major deterrent in wastewater reuse for irrigation. Health risk are associated with pathogens, which may spread diseases through being directly or indirectly ingested into the human body (Dudley et al., 1980; WHO, 1981; 1989; FAO, 1992; Feachem et al., 1983; Shuval, 1991; and Shuval et al., 1986) and fungi (Velez and Diaz, 1985; and Bunes and Merk, 1992).

Pathogens pose the greatest threat to public health; especially when the receiving water is used for domestic recreation on agricultural purpose (Tchobanogeuos, 1979).

The agricultural value of sludge mainly derives from its nutrient content. Sludge, like other organic fertilizers, has long-term beneficial effects on the soil: organic matter contained in sewage sludge improves the physical properties of soil such as aggregate stability, water retention and infiltration, and reduce soil compactibility (Stone et al. 1998). In addition to nutritious content, the organic matter and the C/N ratio are important parameters of the sludge fertilizing potential.

There are many conditions, which may increase the health risk of wastewater reuse in agriculture. The first of these conditions is survival time of pathogenic microorganisms. The natural survival time of pathogenic organisms depends on the carrying medium and the environment. The survival time is a time during which pathogens are capable of causing diseases if they came into contact with a host under favorable condition.

The second of these conditions are  pathogenic bacteria, viruses, protozoa, nematodes and fungi capable of causing diseases which can be found in foods contaminated with sewage water(Bryan,1977; Kowal et al., 1980, and Rosas, 1984). They also can be found harmful to the soil, crops and grazing animals.

On the other hand Pathogenic microorganisms can be transferred from raw sewage and secondary effluent during the irrigation process, directly or in directly to the plants, animal and human, also make various infectious diseases.

Different authors have proved that 5 vegetables are contaminated with microorganisms, when they are irrigated with sewage water and when the soil is fertilized with manure because both usually contain great amounts of pathogenic organisms (Epstein et al., 1982 and Larkin et al., 1978), and when these vegetables are consumed, they could produce diarrhea, salmonellosis, Shigellosis, etc.(Dunlop and Wang, 1961; Kowal et al., 1980 and Rosas et al., 1984).

During the last three decades wastewater reclamation, recycling and reuse in agriculture have received much attention around the world, especially in the arid and semi-arid regions (Neis, 1984; Bouwer, 1982; Emeral and Kayser, 1984; and Madancy, 1981).

Yemen like many other countries in arid and semi-arid regions suffers; from shortage of water resources, so that reuse treated sewage in agriculture is an important question. That is because agriculture seems to be the greatest consumer of water. Annual water consumption has increased dramatically in the last twenty years due to significant social, industrial and agricultural developments. More than 90% of the current water demand is coming from non-renewable groundwater resources in the country.

 Farmers in Yemen, living near the disposal sites of urban wastewater, especially in some of the large cities such as Sana’a, Taiz, Aden, and Ibb are already practicing the reuse of non-treated or partially treated wastewater. (El-Zaemey, 1992). Several countries have produced guidelines, which regulate sewage sludge reuse on the basis of risk to the public health and the environment, however, in Yemen; such guidelines are not established yet.

 In Yemen no investigations have been carried out on the microflora of the sewage and knowledge on the distribution of pathogenic bacteria and fungi in sewage and sludge is absent. Thus, the present study is conducted on the composition, numbers and incidence of various species of bacteria and fungi inhabiting sewage before and after purification.

MATERIALS AND METHODS:

Collection of Sewage Samples:

Thirty samples of each of raw sewage, secondary effluent (500 ml each) and manure (dewatered sludge) (250 gr) were collected from Ibb sewage treatment plant. Each sample was placed in a clean bottle, which was capped tightly and transferred to the laboratory for immediatly bacteriological and mycological analyses.

Five bacterial isolation media were used namely: Nutrient agar for plate count analysis, MF, M-endobroth, MFC agar, MacKonky agar and SS agar. 0.1 ml of each of secondary effluent and raw sewage dilution was used per plate. Three plates of each medium were used for each sewage samples. The counts were calculated per 1ml of sewage, for raw and secondary effluent, and per g dry weight for manure.

Isolation and Identification of bacteria:

Bacteria were encountered using the plate count on nutrient agar.

Total Coliform (TCF) were analyzed using the membrane filtration procedure as described by the APHA (1989) and they were cultured on M-Endo broth (APHA, 1989).

Faecal Coliforms (FC) and faecal Streptococcus (FS) were analyzed using the membrane filtration procedure described by the APHA, (1989). Faecal Coliforms were cultured on M-FC agar (Difco) while faecal Streptococci were grown on m-enterococcus agar (Difco).

        Salmonella concentration was determined using a five tube most probable number (MPN) procedure. Four dilution containing 10^-1/ ml, 10^-2/ml, 10^-3/ml and 10^-4/ ml of raw sludge and Five other tubes for effluent with four dilution containing 10^-1/ml, 10^-2/ml, 10^-3 /ml and 10^-4 ml of secondary effluent were used. Samples were per-enriched in buffered peptone water (BPW) at 37^oC overnight after which 10^-1/mlof per-enrich culture was transferred to Rappaport-Vassilladis broth (RV). Enrichment cultures were incubated at 43^oC and were subcultured to xylose – deoxycolate agar(XLD) after 24 and 48 h. Presumptive Salmonella were purified on MacConky agar without salt and were screened using biochemical and serological tests.

Isolation and Identification of fungi:

        Two isolation media were used for isolation of fungi. Sabouraoud’s dextrose agar (Moss and Mcquown, 1969) containing 40 g/l dextrose, 10 g/l peptone, 20 g agar/l, 40 mg/l Streptomycin, 20 units of Penicillin /ml and 0.05% cycloheximide (Actidione) and Sabouraoud’s dextrose agar containing 40 mg/l Streptomycin and 0.003% rose-Bengal. One ml of the appropriate of each of secondary effluent and raw sewage and manure was used per plate. Three plates were used for each sewage sample. The plates were incubated at 28^oC for 7 days. The counts were calculated per 1ml of sewage.

Identification was carried out by using the taxonomic references of Raper and Fennell (1965), Domsch et al. (1980), Raper and Thom (1949); Ellis (1976); and Moubasher (1993).

RESULTS AND DISCUSSION:

Bacteria recovered from raw sewage, secondary effluent and manure:

The total count of bacteria in the raw sewage, secondary effluent and dewatered sludge were 8.2x 10¹⁰ C/ml, 6.7x 10⁶ C/ml and 5.3x10⁶ C/g respectively (Table 1). The most common bacteria in the above substrates was faecal coliform. It was isolated samples constituting 9.4x10⁸ C/ml, 5.2x10³ C/ml and 4.2x10² C/g respectively. The results in Table (1) show also that the most common bacteria was Escherichia. Coli. It was isolated from all samples of the three substrates constituting 7.6x10⁶, 2.8.x10³ C/ml and 1.2x10² C/g respectively.

Salmonella spp. were isolated from 9 and 3 samples of raw sewage and secondary effluent (2.1x10², 1.3x10 C/ml respectively).

Some authorsreported that the Salmonella spp. can infect or contaminate nearly all living vectors from insects to mammals. (WHO, 1981). Human Salmonella infections and other bacterial infections can be caused from the direct or indirect contact with sewage sludge (Pennsylvania Environmental Network, 2002, WHO, 1981 and Doyle et al., 1997).Most serotypes of Salmonella are pathogenic to humans. A common route of infection for humans is through ingestion of products contaminated with animal faeces (Woolcock, 1991).

 Shigella spp were encountered only from raw sewage (1.1x10 C/ml.). Some bacterial species were also isolated in this study from raw sewage, secondary effluent and dewatered sludge (manure). Some of them can be caused infections directly or indirectly contact with sewage sludge.

 Faecal Streptococcus, Streptococcus pneumonia, Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus cereus were also isolated in the present study from the three substrates. High bacterial count were detected in all samples of the three substrates investigated (Table 1). Faecal coliform bacteria were also detected in high numbers in tested substrates.

Some of these bacteria can produce toxins and cause infections directly or indirectly to human. The great numbers of bacterial colonies were isolated from sewage sludge (raw sewage, secondary effluent and manure, 30 samples of each) at 37°C. The most common bacteria were faecal coliform (Table 1). Simpson (1982) reported that Sewage contain the wide spectrum of Bacteria. The most common bacteria in sewage sludge are the enteric bacteria (Coliforms, Shigellae, Salmonella, etc.). Coliforme bacteria can be contain a rare strain of E.coli that is pathogenic to humans (Kirk, 2003). The typical concentration of E. coli found in untreated sewage sludge is 1000,000 wet weight/g of total solids (Smith, 2003).

The results in this study are analogous to those obtained by several workers in many parts of the world (Kirk, 2003, Smith, 2003 and Simpson, 1982). Results revealed also that the bacterial concentration is high and many of them are Pathogens. Our results in this aspect correspond with those of other authors (Smith, 2003 and WHO, 1981).

Table (1) : Total counts (TC) and number of positive samples (PS) for Bacteria isolated from 30 samples of each raw sewage (colony/ml), secondary effluent (colony/ml) and manure (colony/g).

Fungi recovered from raw sewage:

Sixty five species belonging to twenty three genera were isolated from 30 samples of raw sewage on Sabouraoud’s dextrose agar without (23 genera and 60 species) or with (21 genera and 39 species) cycloheximide at 28 ºC (Table 1). The total number of fungal propagules encountered on both media were 3309.3 and 1404.44 colony per ml.

The most prevalent genera, species on those media were Aspergillus (15), Penicillium (9), Fusarium (5), Cladosporium, (4) Alternaria(4), Cochliobolus (3) Trichoderma(3), Scopulariopsis (2) Mucor (2),Geotrichium (1) and Gibberella (1). They recovered from 66.6 -100% of the samples, constituting 2.17-28.75 % of total fungi respectively. Of the above genera the most frequently encountered species were: Aspergillus flavus, A.fumigatus, A.niger, Acremonium stictum, Aspergillus terreus, A. versicolor, Cladosporium cladosporioides, C. herbarum, C. oxysporum, Gibberella fujikuroi, Cohliobolulus hawaiiensis, Fusarium solani, F. oxysporum, Penicillium chrysogenum, Geotrichum candidum and Scopulariopsis brevecaulis. The above fungi were recovered previously, but with different numbers and frequencies, from sewage and sludge or soil receiving City sewage effluent (Abdel-Hafez and EL-Sharouny, 1987; Abdel-Mallek et al., 1988; Gray, 1982; Niebl, et al., 1982 and Ismail and Abel- Sater 1994).

On Sabouraoud’s dextrose agar with cycloheximide, thirty nine species belonging to sixteen genera were isolated from 30 samples of raw sewage (Table 1).

The total number of fungal propagules encountered on this media was lower compared to those encountered on Sabouraoud’s dextrose agar without cycloheximide (Table 2). The prevalent genera on this media were Aspergillus (9 species), Penicillium (6), Cladosporium, (3), Fusarium (2), Alternaria (2), Cochliobolus (2) Chrysosporium (3), Gibberella (1) and Geotrichum (1). They were isolated from 50-100 of the tested samples. The most common species were: Aspergillus flavus, A. fumigatus, A. niger, Gibberella fujikuroi, Geotrichum and Cohliobolules hawaiiensis were recovered in high frequency of occurrence and constituted 4.60, 3.36, 3.34, 3.35, 3.18 and 2.63 % of total fungi respectively. A, vercicolor, A. ochraceus, Alternaria alternata, A.phragmospora, Acremonium strictum, Cladosporium cladosporioides, C. oxysporum, Fusarium solani, F. pallidoroseum, F. semitectum, Penicillium chrysogenum, P. funiculosum, P. spinolosum, Cochliobolus lunatus, Trichoderma hamatum, T. longibarchiatum, Scopulariopsis brevecaulis and Scopulariopsis brumptii were recovered from 26.6-43.3% of the samples and constituted 1.43-2.93% of total fungi respectively.

Mucor, Rhizopus and Aspergillus tamarrii were not rcovered on this medium, but they encountered on On Sabouraoud’s dextrose agar without cycloheximide in different frequencies. The remaining genera and species were isolated in low or rare frequency of occurrence.

Table (2): Total counts (TC), number of  isolated cases of (NCI), occurrence remarks (OR) and percentage of frequencies (F%) of fungal genera and species recorded from raw sewage on Sabouraud’s agar,

 with and without cycloheximide at 28 ^oC.

Table (2) : Continued

Occurrence remarks (OR), H= high occurrence, from 15-30 cases; M= moderate occurrence, from 8-14 cases; L= low occurrence, from 4-7 cases; R= rare occurrence, from1-3 cases (out of 30 cases).

Fungi recovered from secondary effluent :

Twenty five species belonging to twelve genera were isolated from 30 samples of secondary effluent on Sabouraoud’s dextrose agar with /or without cycloheximide and at 28 ºC (Table 3).

The total numbers of fungal propagules encountered in all samples on both media were 418.6 and 773.3 colony per ml. The prevalent genera on Sabouraoud’s dextrose agar without cycloheximide were Aspergillus (10 species), Fusarium (3), Cladosporium (2) and Penicillium (3) and they were isolated from 100, 66.6, 56.6 and 50% of the samples, constituting 37.5, 12.19, 11.04 and 10.7% of total fungi respectively. The most common species were Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Cladosporium cladosporioides, Gibberella fujikuroi, Fusarium solani, F. oxysporum, Penicillium chrysogenum, and Scopulariopsis brevecaulis. They were recovered from 40-26.6 % of the samples and constituted 6.89- 26.6 % of total fungi. These species were previously recovered but with different incidences from soil receiving city sewage effluent in Egypt (Abdel-Hafez and EL-Sharouny, 1987). The remaining species were collected in low and rare frequency of occurrence. Chrysosporium was not encountered on this media.

The most dominant fungus on Sabouraoud’s dextrose agar containing cycloheximide was Aspergillus (10 species). It was isolated from 100% of the tested samples, constituted 38.17 of total fungi. The most dominant species was A. flavus. It was encountered in moderate frequency of occurrence. The remaining species were isolated in low and rare frequency of occurrence. Cladosporium (2 species), Penicillium (3), Fusarium (3) and Gibbbrella (1). They were isolated in moderate frequency of occurrence and encountered from 43.3, 43.3, 40, and 26.6 % of the total samples, representing 14.2, 13.6, 13.9 and 7.16 of the total fungi respectively.

The remaining genera and species were collected in low or rare frequency of occurrence.

Fungi recovered from dewatered sludge (manure):

Sixty species belonging to 25 genera were isolated from 30 samples dewatered sludge (manure) of secondary effluent on Sabouraoud’s dextrose agar with and without cycloheximide at 28 ºC (Table 4).

The prevalent genera on cycloheximide free medium were Aspergillus (11 species), Penicillium (7), Fusarium (4), Alternaria (3), Cladosporium (3), Trichoderma(3), Cochliobolus (3), Scopulariopsis (3) Mucor (2) and Gibberella (1). They were isolated in high frequency of occurrence, constituting 3.03-28.87 % of total fungi respectively. The most common species were: Aspergillus flavus, A.fumigatus, A.niger, Aspergillus Terreus, Gibberella fujikuroi, Fusarium solani and Penicillium funiculosum. They were recovered from 50-60% of the samples. On the other hand Aspergillus ochraceus, A.versicolor, C. cladosporioides, C. oxysporum, Alternaria alternata, A. tenuissima, Chaetamium globosum, Cohliobolus hawaiiencsis C.lunatus, C. spicifer, Geotrichum candidum, Fusarium oxysporum, Mucor circinoeloides, M. racemosus, Penicillium chrysogenum, Penicillium brevicompactum P. citrinum, Rhizopus oryzae, Trichoderma viride, and Scopulariopsis brevecaulis were collected in moderate frequency of occurrence. They were encountered from 26.6- 46.6% of the total tested samples, constituting 1.42-3.04 % of the total count of fungi. The remaining species were collected in low or rare frequency of occurrence.

 The prevalent genera recovered on Sabouraoud’s dextrose agar with cycloheximide were: Aspergillus (8 species), Penicillium (4), Cladosporium, (2), Fusarium (2), Alternaria(2) and Cochliobolus (2). They were isolated from 50-100 of the tested samples accounting to 30.6- 6.99. % of total fungi respectively. The most common species was: Aspergillus flavus. It was the only species recovered in high frequency of occurrence, constituting 5.26 % of the total fungi.

Aspergillus fumigatus, A. niger, A. terreus, A. ochraceus, Alternaria alternata, Cladospoium cladosporioides, Fusarium solani, Penicillium chrysogenum, P. funiculosum, Cochliobolus lunatus, Scopulariopsis brevecaulis and Gibberella Fujikuroi were recovered in moderate frequency of occurrence (Table 4).

Table (3) : Total counts (TC), number of cases of isolation (NCI), occurrence remarks (OR) and percentage of frequencies (F%) of fungal genera and species recorded from secondary effluent on Sabouraud’s agar,

with and without cyclohexamide at 28 ^oC.

Occurrence remarks (OR), H= high occurrence, from 15-30 cases; M= moderate occurrence, from 8-14 cases; L= low occurrence, from 4-7 cases; R= rare occurrence, from1-3 cases (out of 30 cases).

Table (4) : Total counts (TC), number of cases of isolation (NCI), occurrence remarks (OR) and percentage of frequencies (F%) of fungal genera and species recorded from dewatered sewage (manure)

 on Sabouraud’s agar, with and without cyclohexamide at 28 ^oC.

Table (4) : Continued

Occurrence remarks (OR), H= high occurrence, from 15-30 cases; M= moderate occurrence, from 8-14 cases; L= low occurrence, from 4-7 cases; R= rare occurrence, from1-3 cases (out of 30 cases).

The remaining genera and species were isolated in low or rare frequency of occurrence.

Fungi recovered from the present study have been found in large numbers in sewage (Gray, 1982; Niebl, et al., 1982; and Ismail and Abel- Sater 1994; Diener., et al., 1976; Elland 1981; Larry and Wanger, 1982; Abdel-Hafez and EL-Sharouny, 1987; and Abdel-Mallek et al., 1988).

Also numerous fungi recovered in our study are well known as mycotoxin producing fungi (Tseng, et al., 1995; Aleksandrowies, and Smyk, 1973; Enomoto and Saito, 1972; Pitt, 1994; Rippon, 1982; Sutic, et al., 1979 and Scudamore 1993).

 A certain number of the fungus species recovered in this study are pathogenic (Austwick, 1983; Wadhwani and Srivastava 1985 and Pitt 1994).They are potential facultative causative agents of different mycotic infection (Velzer and Diaz, 1985; Bunse and Merk, 1992, Sutton, et al., 1998, Hoog, et al., 2000).

CONCLUSION:

The workers in the sewage treatment plant most have a health risk during the treatment processes. Health risk are associated with the pathogens, which may spread through being directly, or indirectly ingested into the human body. Pathogens and toxic compounds may be disseminated through Sludge and Sewage, as well as through aerosols (Hickey and Reist, 1975; Bausum et al., 1978 and Bausum et al., 1982). The windy weather raises the question of potential human health hazard passed by pathogen-containing aerosols, in the sewage treatment plant and human communities in the surrounding areas.

The same problem regarding the health of agricultural workers occurs when spray irrigation of sewage effluent is used. Aerosol droplets containing pathogens have been reported to travel up to 1-2 Km (Adams and Spendlove, 1970). Pathogens are more effective when inhaled than when ingested (Melnick, et al., 1978).

Two sewage workers in the Ibb sewage treatment plant suffer from an allergic skin disease (Al-Zubeiry and Al-Shargaby, 1997). But in general sewage workers suffer from an increased incidence of infection or other diseases (Pahren, and Jakubowski,1980; and SWaWWA, 1978). It is important for these workers to have suitable protective clothing, shoes and gloves. Ventilation should be satisfactory, and treatment processed should be automated to the fullest extent possible.

Perhaps the most important single factor is to make sure that sewage workers know how to avoid infection and that they are aware of and use protective measures in their daily work. However, the one of the most important questions, it is the position of sewage treatment plant. Sewage treatment plant must be far away from cities and human communities and must be built in the suitable place from a public health point of view.

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