Impact of Heavy Metals ions on BOD Exertion

Abstract

Abstract Metal Ion Effect In the normal BOD measurement process, microbial matter is taken from some source which is known to be rich in bacteria and capable of undergoing BOD exertion process. It has been established that the type of inoculum becomes important for a study where BOD exertion is measured at different concentrations of heavy metal ions. Wastewaters were digested to grow the bacteria, separately, in Luria broth (LB) medium and then used as seed. Experiments were conducted to determine BOD in presence of some identified heavy metal ions i.e. cobalt, nickel, copper, zinc, silver and cadmium using mixed flora as seed collected from dairy wastewater and distillery wastewater developed in the LB medium. The metal ions were taken in a wide concentration range from 0.2 mM to 10.0 mM to study their effect at different levels of concentrations. A standard mixture of Glucose-glutamic acid (1:1) was taken as food. Industrial wastewater was not used as source of food simply to avoid any inconsistency in the composition of the sample water. Experiments were also conducted to measure optical density at 600nm of microbial medium containing different concentrations of the metal ion. Microbes are expected to show greater optical density in the absence of metal ions while in presence of the metal ions, there is a fall in the absorbance values. The extent of fall depends on the concentration of the metal ions added. In most of the cases, changes in absorbance values correspond to the corresponding changes in BOD. This indicates that the inhibition in BOD is due to the decay of microbial matter. This hypothesis works very well for almost all metal ion environments. Hence, the extent of BOD suppression/ increase is directly related to the decay/ growth of the microbial matter. BOD values were determined in replicas of three bottles for each set of combination of metal ion concentration and by measuring DO levels using membrane based DO meter. Only average values were taken for further calculations. A decrease in BOD is noticed in presence of all the metal ions. For most of the metal ions concentration level upto 1.2mM is good enough to inhibit 100% BOD. However, a presence of zinc ions at these levels results in very less fall in BOD to the extent 5 to 6 % only. Even the larger concentration (10.0 mM) results a decrease in BOD only to the 50%. Percentage change in BOD is plotted as a function of concentration of the metal ion. The presence of silver ions results in a sharp fall in BOD as more than 100 percent inhibition is observed even in the presence of the smallest amount of the metal ion. · An extraordinary toxicity with silver is observed as silver cation forms a more stable complex with sulphur of microbial cell walls. · The solubility product of Ag2S is 6.62x10-50 as compared to for CuS, is 1.28x10-36, which makes silver highly toxic. · The relatively poor toxic behavior of zinc may be due to its chemical nature. Metal ions generally complex through -SH functional groups of enzymes present in the cell wall. Cadmium is more toxic than zinc because the solubility product of CdS is 1.4 x 10-29 while that of ZnS is 2.91 x 10-25. · Mechanisms of cadmium toxicity in microorganisms (not well defined) it enters the cell only by some indirect means There are reports where resistance to cadmium in bacteria is noticed. After showing an increase in inhibition upto 3mM of cadmium concentration, a decrease in inhibition from 35% to 25% is observed. · In the acidic range, suppression is maximum at pH 3.0 for all metal ions except copper. BOD is suppressed to the extent nearly 100% at pH 6.0 except for nickel and zinc. · The increase in BOD with the increase in temperature may be due to greater activity of microbes at higher temperatures. There seems to be significant effect on BOD inhibition due to the presence of metal ion. •The presence of heavy metal ions like cobalt, nickel, copper, zinc, silver and cadmium show behaviour much different when A. odorans is used as a seed in comparison to the wastewater from dairy or distillery. Distillery Wastewater Metal Ion Effect Results of optical density (OD) measurement and percentage change in BOD are presented simultaneously to co-relate change in BOD with that of microbial growth. Out of all metal ion studied, the presence of silver ions is found to be most toxic as also observed for system using distillery waste as a seed. In general, there has been a reasonably good co-relation between the OD value and corresponding %age change in BOD. However, there are indications of resistance being developed towards the presence of metal ions. It is really very difficult to quantify the resistances and co-relation to the concentration levels of metal ions. At the most, it can be linked to the nature of the metal ion. The minimum inhibitory concentration (MIC) of metal ions tolerated by mixed flora from dairy and distillery wastewater through OD and BOD measurements. pH Effect BOD of synthetic samples maintained at different pH values were measured in the absence and presence of each metal ion separately. Experiments were conducted by maintaining a pH in the range 3.0 to 8.0. 2.0mML-1 of the metal ion was maintained in each sample bottle. At this selected concentration, the metal ions exert toxicity to a reasonable extent, as observed. BOD undergoes a substantial decrease to the extent greater than or equal to 100% in the presence of the almost all metal ions. The exception in the observation is in the presence of zinc ions which has already been discussed previously. The metal - microbe complexes are sensitive to change in pH of the medium. Hydrogen ion concentration or pH is probably the single most important factor influencing the metal ion adsorption on both organic and inorganic surfaces. Metal speciation is significantly affected both from hydrooxyl complexation and change in protonation level of the complex. Temperature Effect Results of temperature effect on BOD for both the seeds are similar. BOD is inhibited to the extent 100 to 120 % and there is a little variation with change of the metal ion. At 15°C, BOD exertion does not take place at all which may be due to the reason that microbes are not active at this temperature. BOD Exertion in Presence of Metal Ions using Bacillus brevis and Alcaligenes odorans as Seed

Bacillus brevis Metal Ion Effect In an industrial effluent, the microflora present is of mixed type. Different microbes have different levels of affinities for metal ions and therefore, it is desirable to first isolate and identify each one of all the important colonies that might be present in the effluent. Samples were collected from local sewage treatment plant, Rajpura and pulp & paper industry. Then it was incubated at 37°C for 6 hours for acclimation. 1 mL of each sample is taken for plate count test. A number of colonies are observed on the plates. Important and prominent colonies from both types of wastewaters were isolated and got identified as Bacillus brevis from sewage treatment plant and Alcaligenes odorans from pulp & paper industry. The microbes were cultured in a rich medium and grown at 37°C in incubator. Bacillus brevis and Alcaligenes odorans were used as seed for the BOD measurements. Separate experiments were conducted to monitor the growth of microbes in the presence of different concentration of metal ions added to LB medium by measuring optical density of each system. Measurements were also made in the absence of metal ions. In presence of large concentration of a metal ion, the optical density is decreased. This is true for all the metal ions and indicates that with increase in concentration of the metal ion larger number of microbes are complexed or are mutated and do not contribute towards the optical density values. The minimum inhibitory concentration (MIC) of metal ions tolerated by Bacillus brevis and Alcaligenes odorans are shown in Table 1. Similar metal – microbe complexation processes are taking place in BOD bottles. Results of BOD inhibition are supported by the optical density measurements, i.e., metal-microbe interactions taking outside the bottle. As a result of metal-microbe complexation, the available microbial matter in the BOD bottle is reduced. Lesser the microbial matter, lesser will be the exertion of BOD. Hence, suppression in BOD is observed. Besides other factors like pH, temperature, etc., the magnitude of suppression would also depend on the extent of metal-microbe interactions. Optical density measurements may not give a complete picture of metal – microbe interactions, as in BOD exertion process a sufficient amount of dissolved oxygen is ensured while in optical density measurement, there are no such arrangements. For a given metal ion, the microbial population decreases very fast as observed from optical density values upto a concentration range of 1mM - 3mM (except for silver and cadmium for which the microbial concentration falls at 0.04 mM and 0.5 mM, respectively), depending upon the metal ion. It can be easily inferred that silver shows exceptionally high toxicity towards the microbes. In general, a fall in optical density corresponds to an increase in percentage of inhibition in BOD. These observations lead to the conclusion that Bacillus brevis does form complex with each of the transition metal ion and hence only an apparent BOD is observed. When Ni(II) ions are present in the BOD bottle they form complex with Bacillus brevis and an excellent correlation is seen with the decrease in optical density with the increase in the inhibition as the metal ion concentration increases. In case of Cu (II), there is a sharp decline in growth of the microbes with increase in concentration of metal ion, indicating an extensive metal-microbe interaction. But, the same trend is not observed during BOD exertion as there is a very small or almost negligible inhibition in BOD. In case of zinc ions, the optical density measurements indicate formation of the metal-microbe complex. But the fall in percentage inhibition decreases as the metal ion is added. This trend is in opposition to the expected increase in inhibition with concentration of the metal ion. Results of BOD inhibition in presence of Ag(I) ions show exceptional toxicity for the Bacillus brevis. The optical density measurements also show high degree of toxicity of Ag(I) for Bacillus brevis. Cd(II) forms stable complex with amino acids of microbial cell just like any other transition metal as shown in optical density measurements. This fact is also supported by the fall in BOD exertion in presence of the metal ions, although the concentration levels of these two studies do not correspond as the inhibition in BOD exertion cannot be quantitatively linked to optical density measurements. pH Effect BOD studies were carried out for Bacillus brevis as a seed at different pH 3.0 to 8.0 of the medium. pH of the aqueous medium was maintained by using appropriate amounts of nitric acid or sodium hydroxide. Metal salt buffers were deliberately not used to avoid the introduction of undesirable metal ions in the medium. BOD studies were conducted in presence of 2.0 mML-1 of each metal ion. At pH 7, almost all metal ions inhibit BOD to the maximum. Although no regular trend in BOD inhibition is noticed with change in pH but in the basic range a larger inhibition in BOD is observed in comparison to the acidic medium. In the acidic range, minimum inhibition is observed at pH 5.0. Except for zinc, there is a large decrease in BOD when the pH of the medium is 4.0. It is rather unexpected to observe a greater fall in BOD when pH of the medium is maintained in the basic range (i.e., pH 7 & 8) because metal ions will not be available for complexation with microbes as they are precipitated as metal hydroxides. Temperature Effect The lower temperature is expected to slow down the biochemical process and hence a fall in BOD exertion. Studies on BOD measurement were carried out at different temperatures and the effect of each metal ion was observed. Although BOD exertion is likely to be affected due to changed levels of dissolved oxygen at different temperature, the presence of metal ions do have further influence on the BOD exertion. Results of BOD for the system not containing any metal ion are compared at different temperatures. BOD inhibition due to the presence of the metal ions (2.0 mML-1) of each is also reported. Studies were carried out Bacillus brevis as a seed and GGA as the nutrient medium. For all the metal ions, an increase in BOD exertion when the temperature raised from 20°C to 25°C. A fall in inhibition from 25°C to 30°C and 35°C should be termed as “apparent fall” as this inhibition exists besides the effect of nitrifying bacteria which are known to be most active in the temperature range 30°C to 40°C. The extent of suppression at different temperature does not change with the nature of the metal ions. Almost similar behaviour is observed fall all the metal ions. It was interesting to note the results of experiments for BOD exertion at 15°C. There was practically no change in DO levels at this temperature either in the presence or in the absence of metal ions. It may be change due to the reason that Bacillus brevis are quite dormant below 20°C. In reports, microbial growth is active in the temperature range 25°C to 55°C Bacillus brevis (IMTECH). Alcaligenes odorans Metal Ion Effect Experiments were conducted to measure inhibition in BOD on the addition of different concentration levels of the heavy metal ions. The MIC of metal ions was determined using A. odorans as the microbe (Table 1). Studies were carried out using LB medium in A. odorans. MIC refers to the smallest concentration necessary to inhibit the growth. Thus, lower MIC values indicate more toxic metals and higher MIC values less toxicity. Alcaligenes odorans is a rod like structure and is known to grow in simple nitrogenous environment and is stable upto 42°C. It is an aerobic microbe. Our studies reveal that the presence of heavy metal ions like cobalt, nickel, copper, zinc, silver and cadmium show behaviour much different when A. odorans is used as a seed in comparison to the wastewater from dairy or distillery. The levels of inhibition in BOD are much lower in presence of copper, zinc and cadmium. The bacteria seem to tolerate different concentration levels of metal ions as shown in Table 1. Cobalt and nickel are more toxic, as about 100% decrease in BOD is noticed in presence of 2.0mM and 1.4mM of these metal ions, respectively. Silver is known to be highly inhibitory in its action even at the smallest concentration of it. Different levels of tolerance of metal ions may be due to the different complexing nature and their different biochemical preferences for the respective metal ions. It can also be due to the different electrochemical nature of metal ions. The metal ions with high reduction potential shall preferably get reduced and will be less toxic for the microbe. pH Effect Effect of metal ions on BOD exertion was carried out at different pH values of the medium in the range 3-8. The results indicate that the presence of metal ions significantly decrease the BOD. Each metal ion behaves in a different way because of its different chemical stability at different pH values in the range 3 – 6. Results indicate that inhibition in BOD is not affected by change in pH in the range 6 – 8, probably, because of similar chemistry of heavy metal ions in this pH range. In acidic medium, all the metal ion species behave in a similar nature at pH = 3. Temperature Effect As discussed in previous chapters, the effect of temperature on the extent of inhibition/ increase in BOD is very complicated. This involves a change in BOD due to the presence of heavy metal ions at different temperatures as well as different concentrations. This change has to be viewed in the background of different DO levels at different temperatures. In the present studies it was observed that BOD exertion does not take place at all at 15°C to 20°C. From the studies at temperatures 25°C, 30°C, 35°C, BOD inhibition takes place almost to the same extent for all the heavy metal ions used. The presence of the metal ions leads to an inhibition of 120% at 25°C, 30°C, 35°C. It can be due to the similar chemical ions which do not change with temperature in this range. In reports, microbial growth is active in the temperature range 25°C to 45°C for Alcaligenes odorans.

*** SEED Metal ion concentration (mM) Co2+ Ni2+ Cu2+ Zn2+ Ag+ Cd+ Dairy 1.2 1.2 1.2 >10.0 0.2 4.0 Distillery 10.0 0.8 8.0 - 0.2 - B. brevis >14.0 6.0 10.0 8.0 - 0.2 A. odorans 3.0 2.0 9.0 7.0 0.2 10.0