Enhancement of bacterial efficiency for metal removal using mutation techniques

The objective of the present study was to obtain by mutation and selection techniques bacterial strains capable of removing heavy metals at high efficiency. Four of the bacteria most promising in metal uptake, Staphylococcus aureus, Bacillus Sphaericus, B. licheniformis and Arthrobacter sp. were selected after isolation from water heavily polluted with heavy metals. Two mutagenic agents were used: U.V. irradiation at 245nm (physical) and 1% ethidium bromide (chemical). Optimum conditions for metal removal by most of the tested bacteria were: pH 9, 50°C and 200rev/min agitation speed. Induction of mutation both physically or chemically resulted in mutants that were superior over their wild types in removing heavy metals under investigation. The highest removal efficiencies (REs) achieved were in the following order: Cd(89.9–100%); Cr(87.3–99.7%); Zn(47.7–100%); Cu(40.8–84.7%); Pb(40.2–51%); Fe(17.5–28.7%); Ni(13.8–23.9%) and finally Co(17.2–18.4%). Using mixed cultures of the wild and the selected mutants enhanced the RE(s) of some metals compared to those obtained by individual species, and the time required to achieve the highest RE was reduced.     

Enhancement of alkaline protease production in Bacillus circulans using plasmid transformation

Plasmid transformation is an efficient and crucial biotechnological tool that enables the enhancement of many important microbial characters that would be beneficial in a lot of industrial, agricultural and environmental applications. In the present study, five Bacillus species (B. subtilis, B. cereus, B. alvei, B. circulans and B. pumilus) were investigated. They were isolated from agricultural soils of different local arid environments of the Kingdom of Saudi Arabia, identified and characterized for their plasmid content. The main objective of the present study was to enhance the production of alkaline protease in Bacillus circulans (the recipient strain) through plasmid transformation from B. subtilis (the donor strain). All the tested Bacillus strains successfully produced unique multiple (3, 4 and 5) spontaneous antibiotic resistant mutants against chloramphenicol, neomycin, rifampicin, streptomycin, kanamycin and tetracycline and all of which were mutated to Rif_r strains. B. pumilus showed the highest resistance against five of the six tested antibiotics while both of B. alvei and B. circulans showed the lowest resistance to only three of the tested antibiotics. Results revealed that B. subtilis was the best among the tested species concerning the production of alkaline protease (90.2 U/ml) while B. pumilus was the lowest in activity (40.3 U/ml). Screening of plasmid content revealed the presence of one or two mega indigenous plasmids in all the tested species. The four transformant strains BC ₁ , BC ₂ , BC ₃ and BC ₄ resulting from plasmid transformation exhibited significant increases in the activity of alkaline protease and recorded 2.31- to 3-fold increases compared to the parent B. circulans cells and 2.11- to 2.75-fold increases compared to the donor cells of B. subtilis. They also acquired antibiotic resistance to tetracycline and chloramphenicol that was completely absent in the parent cells of B. circulans. Results revealed that plasmid transformation among the tested Bacillus spp. is a powerful technique that can be efficiently exploited to enhance alkaline protease production in the transformed Bacillus spp. compared to their wild strains and we recommend using the improved transformant strains for commercial and industrial purposes.     

Enhancement of nodulation by some arid climate strains of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.     

Bioremediation of Vegetable Oil and Grease from Polluted Wastewater Using a Sand Biofilm System

Pseudomonas sp. (L1), P. diminuta(L2) were among eight bacterial strains isolated from vegetable grease and oil-contaminated industrial wastewater, four of which only were found to have the ability to degrade oil and grease. They were identified and investigated for oil and grease degradation either individually or in combinations in previous unpublished work by the authors. Since the combination M1 (Pseudomonas sp. andP. diminuta) produced the highest degradative activity, it was used in the present study in a biofilm sand filter system for vegetable oil and grease removal. This system was tested either as one unit or two units in sequence where different flow rates (30, 50, 100 ml/h) were applied compared to a control unit(s). Results showed that both biofilm systems reduced oily wastewater, even in cases of high degree of pollution (fat, oil & grease (FOG), 7535 ppm; biochemical oxygen demand (BOD₅), 525 ppm; chemical oxygen demand (COD), 1660 ppm). Results also showed a removal of FOG with efficiency at 100%; BOD₅ at 95.9% and COD at 96%, at 50 ml/h flow rate using one unit of biofilm system. On using two units in sequence, a complete removal of FOG, BOD₅ and COD with efficiency 100%, at flow rate 100 ml/h was achieved. In conclusion, the previous biofilm results indicated the efficiency of such a system in treating oily polluted wastewater (vegetable oil origin) on the basis of bacterial isolates being used, the optimum flow rate, and the number of biofilm units used in sequence to obtain the highest removal capacity of such a system. This revised version was published online in July 2006 with corrections to the Cover Date.     

Treatment of mixed domestic–industrial wastewater using cyanobacteria

Alexandria Sanitary Drainage Company (ASDCO), Alexandria, Egypt has two primary treatment plants, the eastern and the western wastewater treatment plants (EWTP and WWTP) that receive mixed domestic–industrial influents and discharge into L. Mariut. The lake is subjected therefore to severe levels of pollution and dominated by members of cyanobacteria that can cope with the high pollution load in the lake water. Isolation and utilization of the locally generated cyanobacterial biomass for remediation processes of highly toxic pollutants offers a very efficient and cheap tool for governmental or private industrial activities in Alexandria and will generate a source of revenue in Egyptian localities. The main objective of the present study was to investigate the biodegradation and biosorption capacity of some potential cyanobacterial species dominating the lake ecosystem toward organic and inorganic contaminants polluting the primary-treated effluents of the EWTP and WWTP. The primary effluents were subjected to biological treatment using three axenic cyanobacterial strains (Anabaena oryzae, Anabaena variabilis and Tolypothrix ceytonica) as batch system for 7 days. Removal efficiencies (RE) of the different contaminants were evaluated and compared. Results confirmed the high efficiencies of the investigated species for the removal of the target contaminants which were species and contaminant-dependent. BOD₅ and COD recorded 89.29 and 73.68% as maximum RE(s) achieved by Anabaena variabilis and Anabaena oryzae, respectively. The highest RE of the TSS recorded 64.37% achieved by Tolypothrix ceytonica, while 38.84% was recorded as the highest TSD RE achieved by Anabaena variabilis. Tolypothrix ceytonica also exhibited the highest RE for FOG recorded 93.75%. Concerning the contaminant metals, Tolypothrix ceytonica showed the highest biosorption capacity where 86.12 and 94.63% RE were achieved for Zn and Cu, respectively. In conclusion, results of the present study confirmed the advantageous potential of using the tested cyanobacterial species for the treatment of contaminated wastewater. Results also clearly showed the quality improvement of the discharged wastewater which in turn will eliminate or at least minimize the expected deterioration of the receiving environment.     

Pollution control in pulp and paper industrial effluents using integrated chemical–biological treatment sequences

The main objective of the present study was to improve the quality of pulp and paper industrial wastewater of two local mills RAKTA and El-Ahlia, Alexandria, Egypt, and to bring their pollutant contents to safe discharge levels. Quality improvement was carried out using integrated chemical and biological treatment approaches after their optimization. Chemical treatment (alum, lime, and ferric chloride) was followed by oxidation using hydrogen peroxide and finally biological treatment using activated sludge (90 min for RAKTA and 60 min for El-Ahlia effluents). Chemical coagulation produced low-quality effluents, while pH adjustment during coagulation treatment did not enhance the quality of the effluents. Maximum removal of the tested pollutants was achieved using the integrated treatment and the pollutants recorded residual concentrations (RCs) of 34.67, 17.33, 0.13, and 0.43 mg/l and 15.0, 11.0, 0.0, and 0.13 mg/l for chemical oxygen demand (COD), biochemical oxygen demand (BOD₅), tannin and lignin, and silica in RAKTA and El-Ahlia effluents, respectively, all of which were below their maximum permissible limits (MPLs) for the safe discharge into water courses. Specific oxygen uptake rate (SOUR) and sludge volume index (SVI) values reflect good conditions and healthy activated sludge. Based on the previous results, optimized conditions were applied as bench scale on the raw effluents of RAKTA and El-Ahlia via the batch chemical and the biological treatment sequences proposed. For RAKTA effluents, the sequence was as follows: (1) coagulation with 375 mg/l FeCl₃, (2) oxidation with 50 mg/l hydrogen peroxide, and (3) biological treatment using activated sludge with 2,000 mg/l initial concentration and 90 min hydraulic retention time (HRT), while for El-Ahlia raw effluents, the sequence was (1) coagulation with 250 mg/l FeCl₃, (2) oxidation with 45 mg/l hydrogen peroxide, and (3) biological treatment using activated sludge with 2,000 mg/l initial concentration and 60 min HRT. In conclusion, results confirmed that the application of the proposed sequential treatments removed almost all COD, BOD₅, high molecular weight compounds, and silica from RAKTA and El-Ahlia influents and produced high-quality effluents, thus achieving the main objective of this study.     

Toxicity and biodegradation of fluometuron by selected cyanobacterial species

The Biodegradation capabilities of six selected cyanobacterial species for fluometuron, a phenylurea herbicide, as well as its inhibitory effect on chlorophyll a content were investigated. The selected species (three strains of Microcystis aeruginosa, Anabaena cylindrica, A. flos-aquae and A. spiroides) were subjected to three elevated concentrations of fluometuron (0.14, 0.7 and 1.4 mg/ml) for different exposure times (1–5 days). Results revealed that biodegradation of fluometuron is species-dependent and positively correlated with the exposure time, reaching maximum efficiency after 5 days at all the investigated concentrations. All the species tested showed generally great ability to degrade the compound even at the highest concentration with specific variations among them. Biodegradation efficiencies of fluometuron by the selected species were in the following ranges; 39.2–99.9; 87.5–100; and 93.2–100 at 0.14; 0.7 and 1.4 mg fluometuron/ml respectively. It was noticed that the gradual increase in the pesticide concentration enhances its biodegradability by the selected algal species. Variations according to species as well as exposure time were discussed. The highest fluometuron concentration (1.4 mg/l) showed the highest inhibition of chlorophyll a content in the tested species and toxicity was also species- and time-dependent.     

Comparison between biological and chemical treatment of wastewater containing nitrogen and phosphorus

The present work compared chemical and biological treatment methods to achieve the most efficient treatment for the reduction or elimination of phosphorus and nitrogen from mixed industrial–domestic wastewaters. Batch chemical precipitation by ferric chloride and aluminum sulfate (alum) and a continuous biological suspended growth system were investigated as well as the optimum operating conditions. Concerning chemical treatment, Alum generally achieved a higher removal efficiency percentage for the investigated pollutants compared with FeCl₃ at their optimum pH and dose, especially with chemical oxygen demand (COD). FeCl₃ treatment achieved success only with phosphorus removal, while none of the COD, 5-day biochemical oxygen demand (BOD₅), total nitrogen (TN) and N–NH₃ achieved acceptable treatment and remained above the maximum permissible limits (MPL). Thus, for such wastewaters, alum is more efficient than FeCl₃. Biological treatment exhibited higher efficiencies, particularly towards nitrogen. TN removal increased by increasing the flow rate to 30–60 l/day. N–NH₃ removal was effective at the slowest flow rate and decreased with increasing flow rate, while an opposite trend was recorded for N–NO₃. At all flow rates, phosphorus levels were below the accepted MPL for discharging into natural systems. Moreover, there was a general trend for the proposed biological treatment to achieve a high removal efficiency for BOD₅ and COD, bringing them to acceptable levels to be released into watercourses safely, especially at the slowest flow rates. Thus, integration between the proposed chemical and biological treatment is highly recommended, producing high-quality effluents acceptable by the environmental law.     

The Potentiality of Free Gram-negative Bacteria for Removing Oil and Grease from Contaminated Industrial Effluents

Summary Eight bacterial species were isolated from vegetable oil and grease-contaminated industrial wastewater, only four of which were found to have the ability to degrade oil and grease in the contaminated wastewater. These isolates were identified according to morphological and biochemical profiles as, Pseudomonas sp. (L1), P. diminuta (L2), P. pseudoalcaligenes (L3), and Escherichia sp. (L5). The degradative capabilities of the identified bacterial isolates for Tween 20 (Tw20) were investigated under different pH levels (6.5, 7, 7.5, and 8), different temperatures (30 and 37 °C) and different concentrations of Tw20 (1, 1.5, and 2%). Results revealed differences in their optimum conditions for maximum degradation of vegetable oil. Bacterial isolates were tested individually or in combinations using synthetic aqueous medium supplemented with 1% palm oil, incubated at 30 °C, and agitated at 150 rev/min for 13 days. All the tested bacteria were able to degrade the palm oil completely and utilized the free fatty acids (FFA) as a carbon source. The combination M1 (Pseudomonas sp. and P. diminuta) produced the highest degradative activity, followed by M3 (Pseudomonas sp., P. diminuta and P. pseudoalcaligenes). Also M1 produced the highest activity in reducing COD (93%) and BOD₅ (100%).