Based on experimental work, the effect of pH on heterotrophic bacteria enumeration was
studied in batch cultures tests conducted under different chromium concentrations. Samples were
collected from the effluent of the primary facultative pond of the University of Dar es Salam (Tanzania)
waste stabilization ponds system. The enumeration of heterotrophic bacteria was investigated at different
pH values. The density of bacteria was higher at pH 7 than any other pH values for all concentrations. At
this pH, the number varied between 1,450,000 to 280,000 at 12 and 108 hours after addition of 5 mg/l
chromium concentration. In case of 50 mg/l and pH 7 the number reduced dramatically to 98 to 0 at 12
and 108 hours respectively. It was observed that, the pH of 11 was significantly detrimental to the
heterotrophic bacteria. The number ranged between 4000 to 500 in case of 5 mg/l after 12 and 108 hours.
Only 13 heterotrophic bacteria were counted after 12 hours when 50 mg/l of chromium concentration was
added

Ali, Yousif

Gamiel, Masoud

English

SUST Journal Systems (Sudan Univ. of Science and Technology)

SUST Journal of Engineering and Computer Science (JECS), Vol. 15, No. 1, January 2014  11  Effect of Chromium on the Survival of Heterotrophic Bacteria in Waste  Stabilization Ponds  Masoud Gamiel Ahmed1 and Yousif Ali Yousif2  School of Civil Engineering, Sudan University of Science and Technology (SUST)1 College of Water and Environmental Engineering, Sudan University of Science and Technology (SUST)2 masoudadli@yahoo.com  Received:01.07.2013 Accepted:11.09.2013  ABSTRACT-Based on experimental work, the effect of pH on heterotrophic bacteria enumeration was studied in batch cultures tests conducted under different chromium concentrations. Samples were collected from the effluent of the primary facultative pond of the University of Dar es Salam (Tanzania) waste stabilization ponds system. The enumeration of heterotrophic bacteria was investigated at different pH values. The density of bacteria was higher at pH 7 than any other pH values for all concentrations. At this pH, the number varied between 1,450,000 to 280,000 at 12 and 108 hours after addition of 5 mg/l chromium concentration. In case of 50 mg/l and pH 7 the number reduced dramatically to 98 to 0 at 12 and 108 hours respectively. It was observed that, the pH of 11 was significantly detrimental   to the heterotrophic bacteria. The number ranged between 4000 to 500 in case of 5 mg/l after 12 and 108 hours. Only 13 heterotrophic bacteria were counted after 12 hours when 50 mg/l of chromium concentration was added.    Keywords: Chromium, Heterotrophic Bacteria, pH  7ا9:;<=>- ?@A7ا ABCDE FGارد J>E ،FB:>L>7ا برNO<7ا P:Q ادN><QأBSBTورVBW7ا7NW PSBTورVB NXAB<YNZ7ا دVQ P:Q7اW FB[\وA<B موA[7ا د^Tو P\F_:<;` aBYاA<b .راد FL`NT P\ تN_:;>7ا FO7NL>7 FB7وdا ةABfZ7ا جA;` h` تNSBL7ا JL>T NBiاaSE ،مj=7ا . ?B@ VSQ هدNOXأ ?E يAB<[Z7ا داVL<7ا>@A:7 F_:<;`PSBTورVBW7N . ?@A7ا VSQ NXAB<[Z:7 دVQ P:Qأ نأ VTوPSBTورVBW7ا 7 موA[7ا aBYاAE n:<;` P\ . hBb دVL7ا حواA<X  ?@A7ا اpھ VSQ1,450,000  P7ا280,000  VLb12  و108   F\Nrأ h` FQNG5?O:`/ sBEA<7ا P:QA<7.  F\Nrأ F7Nt P\50 ?O:`/ ?@رو موA[7ا h` A<7ھB PSBTورVB7  P7ا يAB<[Z7ا دVL7ا 9@NS<X98  و0  VLb12 و108 sBEA<7ا P:Q موA[7ا  F\Nrأ h` FQNG . 9@NS<X يAB<[Z7ا دVL7ا نأ utjE Vv7 ?@ر VSQ ةVwbھ PSBTورVB11 . hBb ?@A7ا حواAE4000  P7ا500  F\Nrأ F7Nt P\5 ?O:`/7ا h` A<7 VLb موA[12 و108   F\Nrأ h` FQNGموA[7ا . xv\13  VLb تVTو NXAB<[Z7ا h`12  F\Nrأ h` FQNG50 ?O:`/موA[7ا h` A<7.  INTRODUCTION Chromium is a metallic element that functions as a co-factor to augment the action of insulin, and thus influences carbohydrate, protein, and fat metabolism. It is odorless and tasteless and occurs naturally in various types of rock, soil, ore and volcanic dust as well as in plants, animals and humans. The most common forms of chromium are trivalent (chromium 3) and hexavalent (chromium 6); In the environment, chromium usually exists in the trivalent form. It is possible for one form to change into the other when chemical conditions are right. Hexavalent chromium is recognized as a human carcinogen when it is inhaled. Chronic inhalation of hexavalent chromium has been shown[1] to increase risk of lung cancer and may also damage the small capillaries in kidneys and intestines.   Other adverse health effects associated with hexavalent chromium exposure, according to the National Institute for Occupational Safety and Health (NIOSH), include skin irritation or ulceration, allergic contact dermatitis, occupational asthma, nasal irritation and ulceration, perforated nasal septa, rhinitis, nosebleed, respiratory irritation, nasal cancer, sinus cancer, eye irritation and damage, perforated eardrums, kidney damage, liver damage, pulmonary congestion and edema, epigastric pain, and erosion and discoloration of one's teeth[1].   SUST Journal of Engineering and Computer Science (JECS), Vol. 15, No. 1, January 2014  12  Microorganisms play an important role in biological treatment. Microorganisms have been responsible for the natural stabilization of human and animal wastes since the origin of these species on earth [2]. Heterotrophic bacteria are the most important species in the degradation of organic materials. It can be further classified as aerobic, anaerobic or facultative, depending on their need of oxygen. Wastewater containing chromium with high concentration, discharged into sewer system without pretreatment can cause adverse effects on biological wastewater treatment processes. Therefore, only low concentration of chromium is allowed in the effluent discharged to receiving waters, because of toxicity to aquatic and human life.   Since most municipal wastewater treatment facilities use biological treatment processes for stabilization of organic and inorganic substances in wastewater, it is necessary to have knowledge of the effect of chromium on these processes. Therefore, it is important to understand the effect of chromium on the toxicity to heterotrophic bacteria in order to limit its concentration in industrial effluents which are treated in waste stabilization ponds.  Chromium enters the waste stream generally in the form of hexavalent salts such as sodium chromate, potassium chromate, and sodium dichromate.   These salts are used extensively in industries, such as electroplating, paint and pigment manufacturing, textile, fertilizer and leather tanning[3]. Most industrial wastewater must first pass through a sewage treatment plant. Since bacteria in such plants are destroyed by trace amount of chromium, the treatment plant becomes no longer effective. Temperature and pH play a vital role on survival and growth of bacteria. In general, optimal growth occurs within a fairly narrow range of temperature and pH, although the bacteria may be able to survive within much boarder limits. The objective of this paper is to study the effect of chromium on the survival of heterotrophic bacteria in waste stabilization ponds. MATERIAL AND METHODS Sampling To obtain reliable results from any bacteriological analysis of water samples, one has to strictly follow the standard procedures laid down for their collection, dispatch to the laboratory, storage and sample examination. In this paper, all laboratory examinations were carried out in accordance with Standard Methods[4].APHA, AWWA and WPCF. Sampling Apparatus  Cleanness of accessories used can affect the quality of samples and finally the results from the analysis. Sampling bottles for microbiological examination where cleansed and rinsed carefully and were given a final rinse with distilled water. All of the equipment used for the examination of heterotrophic bacteria were sterilized for more than two hours at 1700o C in a hot air oven before collection of samples. Sampling Procedure To assess the effect of chromium on survival of heterotrophic bacteria in waste stabilization ponds, grab waste water samples were collected from the effluent of the primary facultative pond of the University of Dar es Salaam waste stabilization ponds system. A beaker was used to collect samples and the content was poured into six wide mouth glass bottles of 500 ml capacity each. An ample air space was left in the bottle (at least 2.5 cm) to facilitate mixing by shaking, prior to examination. Preparation of chemicals Preparation of chromium  To prepare solution to be used in the tests, 141.45 mg of K2Cr2O7 was dissolved in 1000 ml of distilled water to make 50 mg/1 solution of chromium ions. From the 50 mg/1 of chromium solution, different dilutions of 5 mg/1, 10 mg/1, 15 mg/l, 25 mg/1 and 50 mg/1 were prepared by making use of the heavy metal solutions and the samples. Preparation of Agar The agar was prepared according to the specification given by the manufacturers. 35 gm of agar was diluted in 1.0 liter of the prepared distilled water. The agar was then sterilized in an autoclave for 15 minutes at a temperature of 121° C and a pressure of 1.06kg/cm2. The SUST Journal of Engineering and Computer Science (JECS), Vol. 15, No. 1, January 2014  13  prepared agar was left to cool down to a temperature of about 40°C in an incubator adjusted to that temperature before pouring into the plates so that it would not kill the bacteria. Bacteriological Analysis The effect of chromium on the survival of heterotrophic bacteria in waste stabilization ponds was investigated in batch cultures. Samples were collected from the effluent of primary facultative pond. Chromium ions were added artificially. Samples were adjusted to pH 7 and were kept at room temperature during the determination of the effect of chromium at varying concentrations. The second run was done to find the influence of chromium concentrations at fixed pH of 4, 6, 7, 8, 9 and 11 and the samples were kept at room temperature. In this study pour plate count was used to enumerate the heterotrophic bacteria because of its ability to give best information on the number of bacteria. Two replicates in appropriate dilutions were used. Dilution water was prepared from distilled water, presterilized in an autoclave and allowed to cool at room temperature. By using pipettes, 1.0 ml of each diluted sample (plus the heavy metal ions, except for the control sample) was placed in Petri dishes.  The time lapse between making the dilutions and pouring the plates was less than 20 minutes. Then about 15 ml or the liquefied agar medium was poured in each petri dish containing the sample. The agar and sample was thoroughly mixed together for a uniform dispersion of organisms by gently tilting and rotating the dish, taking care not to splash upper portion. The mixture of diluted samples with agar was then left for about 15 minutes to allow agar to harden on a level surface. After making sure that agar has hardened enough, the plates were removed and incubated at 35°C for 72 hours in accordance with Mayo [5].  After the incubation period, colonies on the petri dishes were counted. The new samples were reincubated after every 24 hours. The duration of the whole experiment was 96 hours.  The effect of chromium on heterotrophic bacteria at varying pH The sample was divided into thirty six portions of 300 ml each (beaker). Out of the thirty six portions of samples, six groups were selected. Each group contained six equal volume portion of sample of 300 ml. Each group was dosed with different concentration of chromium (0, 5, 10, 25 and 50 mg/1) and in each single group each sample was kept at constant pH of 4, 6, 7, 8, 9 and 11. Samples were adjusted artificially to the desired pH by addition of either NaOH or H2SO4 for raising or lowering pH, respectively. Analysis of heterotrophic bacteria count was done as explained.  RESULTS AND DISCUSSION Tables 1 to 6 show the results of the effect of the pH on the enumeration of heterotrophic bacteria. The density of heterotrophic bacteria was higher at pH 7 than any other pH value for all concentrations. Table1shows the effect of pH on heterotrophic bacteria after 12 hours from chromium injection. The number ranged between 145,000 to 98 at pH 7 at concentration 5 to 50 mg/l, respectively. The pH may have a significant role on the processes associated with the change of the state of chromium ions and hence on the decay of heterotrophic bacteria. The most adverse effect of pH was observed at pH 11, the number varied between 4000 to 13 at concentrations 5 and 50 mg/l.  Table 2 shows the trend of heterotrophic bacteria count after 36 hours. Again the trend shows the mortality rate of heterotrophic bacteria is minimum at pH 7, which confirms that the optimum pH for bacteria growth is near neutral pH. Similar trend observation have been recorded but with less number of bacteria when count conducted after 60, 84 and 108 hours from chromium injection. The number goes less as time increased (Tables 3, 4 and 5) .No bacteria have been counted after 36 hours for higher concentrations( 25 and 50 mg/l). The mortality rate for control sample (without chromium) was found to be higher at pH of 11. Significant mortality rate of bacteria was also observed at pH 4 and 6. This shows that pH fluctuation in waste stabilization ponds may affect the population of heterotrophic bacteria SUST Journal of Engineering and Computer Science (JECS), Vol. 15, No. 1, January 2014  14  even in absence of heavy metals such as chromium. Table 6 shows the mortality rate of heterotrophic bacteria at various pH values and concentration of chromium. For a given dose of chromium concentration, the mortality rate was minimum at pH 7. Likewise for the given pH, the mortality rate of heterotrophic bacteria increased as chromium concentration increased.  CONCLUSIONS The influence of pH on the heterotrophic bacteria growth was investigated at different chromium concentrations. The optimal pH value for bacteria was found to be 7. Significant mortality rate of bacteria was observed at pH 4 and 11. The mortality rate of heterotrophic bacteria was high at pH 4 and 11 even in absence of heavy metals such as chromium.  REFERENCES [1] http://www.cdc.gov/niosh/topics/chromium/ [2] Okun, D.A. and Poghis, G. (1975) Community wastewater collection and disposal. World Health Organization, Geneva. [3] Ganguli, A. and A.K. Tripathi (2002), “Bioremediation of toxic chromium from electroplating effluent by chromate-reducing Pseudomonas aeruginosa in two bioreactors” Appl. Microbiol. Biotechnol., 58: 416-420. [4] American Water Works Association), Water Environment Federation and E.W. Rice (2012), Standard Methods for the examination of water and wastewater 22ndEdition. American Public Health Association.  [5] Mayo, A. W., (1997) “Response of mixed cultures of Chlorella vulgaris and heterotrophic bacteria to variation of pH”,Wat. Sc.Tech., Vol. 30,No.8, pp. 285-294.  Table 1: Effect of pH on the enumeration of heterotrophic bacteria count after 12 hours pH Number of heterotrophic bacteria at different concentrations  0 5 10 15 25 50 pH=4 1020000 82000 7000 2200 460 42 pH=6 1550000 125000 10500 3500 770 59 pH=7 18000000 145000 122000 38000 9000 98 pH=8 14300000 1180000 98000 32000 7400 87 pH=9 6200000 500000 42000 13000 3100 72 pH=11 49000 4000 350 100 22 13  Table 2: Effect of pH on the enumeration of heterotrophic bacteria count after 36 hours pH Number of heterotrophic bacteria at different concentrations  0 5 10 15 25 50 pH=4 460000 28000 3000 700 46 0 pH=6 640000 43000 4500 1100 76 0 pH=7 4500000 500000 53000 8700 900 49 pH=8 3500000 390000 43000 8000 730 30 pH=9 1500000 171000 18000 4000 290 13 pH=11 22000 1300 145 40 0 0   Table 3: Effect of pH on the enumeration of heterotrophic bacteria count after 60 hours pH Number of heterotrophic bacteria at different concentrations 0 5 10 15 pH=4 140000 25000 1300 500 pH=6 200000 38000 1900 800 pH=7 2400000 440000 22000 7600 pH=8 2000000 360000 17800 6200 pH=9 850000 152000 7600 3000 pH=11 7000 1200 62 30  SUST Journal of Engineering and Computer Science (JECS), Vol. 15, No. 1, January 2014  15  Table 4: Effect of pH on the enumeration of heterotrophic bacteria count after 84 hours pH Number of heterotrophic bacteria at different concentrations  0 5 10 15 25 pH=4 128000 12000 1000 400 23 pH=6 192000 18000 1500 600 39 pH=7 2000000 360000 16600 5400 450 pH=8 1800000 290000 13400 4400 370 pH=9 770000 73000 5700 2000 159 pH=11 6000 600 47 20 0 Table 5: Effect of pH on the enumeration of heterotrophic bacteria count after 108 hours pH Number of heterotrophic bacteria at different concentrations  0 5 10 15 25 pH=4 101000 9000 720 300 300 pH=6 153000 14000 1200 500 500 pH=7 1800000 280000 12000 4200 4200 pH=8 1600000 175000 9800 3300 3300 pH=9 600000 62000 4300 1700 1700 pH=11 5000 500 40 18 0 Table 6: Mortality rate of heterotrophic bacteria at varying pH Conc. of chromium (mg/1) Mortality rate (k) (hr-1) 4 6 7 8 9 11 0 0.2818 0.2469 0.0426 0.0617 0.1314 0.5348 5 0.4919 0.4567 0.2525 0.2696 0.3412 0.7436 10 0.6969 0.6631 0.487 0.477 0.5476 0.9466 15 0.7934 0.7547 0.5559 0.5703 0.6453 1.05 25 0.9052 0.8620 0.6574 0.6737 0.7462 1.1585 50 1.1047 1.0763 1.034 1.044 1.0597 1.2024          

Effect of Chromium on the Survival of Heterotrophic Bacteria in Waste Stabilization Ponds

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Effect of Chromium on the Survival of Heterotrophic Bacteria in Waste Stabilization Ponds

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