Removal of Fluoride Using Pseudomonas Putida

Water is an essential resource for sustaining life. Over the past few decades, the ever growing population, urbanization, industrialization and unskilled utilization of water resources have led to degradation of water quality in various developing countries. Presence of various hazardous contaminants like fluoride, arsenic, nitrate, and sulfate and other heavy metals, etc. in underground water has been reported from different parts of world including India. Fluoride uptake of up to mg/L has beneficial effects on plants, animals, and human beings. In human, fluoride exhibits bone formation, remineralization and protects against demineralization of teeth. Fluoride is extremely effective in protecting cavities and making teeth stronger. However, excessive fluoride intake over a long duration of time may result in a serious health problem called fluorosis. The starting phase is known to be dental fluorosis, which results in dental deformities with the development of a yellow colour affecting children in the age range of 6-8 years. The next phase is skeletal fluorosis, which brings the fluoride deposition in skeletal muscles in children as well as adults. The most alarming stage of fluorosis is crippling fluorosis, which can have life threatening effects. Considering the health impacts of fluoride, World Health Organization (WHO) has set a fluoride concentration limit of 1.5 mg/L for drinking water where the limit of fluoride concentration set by the Central Pollution Control Board (CPCB) is 2.0 mg/L for inland surface water [1].

Fluoride enters a groundwater stream through natural as well as anthropogenic sources such as deep percolation from intensively cultivated fields, disposal of hazardous wastes from industry, liquid solid wastes from industries, sewage disposal, surface impoundments, etc. Some important industries that releases fluoride into a water stream are steel, aluminium, copper, and nickel production, phosphate ore processing, phosphate fertilizer production, glass, brick, and ceramic manufacturing, etc [2].  Fluoride concentrations in different industrial effluents may vary; in the effluent of some industries like phosphatic fertilizer; the fluoride concentration is normally around 20 mg/L. In many cases, the concentration of other ions associated with fluoride-containing effluents is below their permissible limits. To reduce the chance of contamination of groundwater by these industries’ effluents, the removal fluoride from the wastewater stream is essential before it enters the surface water stream and pollutes groundwater. Important methods used for fluoride removal are distillation, membrane separation processes, precipitation, ion exchange, and adsorption[3].

The adsorption method entails the use of physical or the biological adsorbents. The biological treatment method involving living microbial cells evolved as an emerging field for the removal of pollutants from water due to its low cost and eco-friendly nature. Further, in biological removal methods, microorganisms may be used in either a bulk phase or immobilized phase [4,5].Immobilization can improve the microbial process’s efficiency. Some microbial strains have been used to remove fluoride from water in the bulk phase as well as immobilized phase. Many microbial strains have also been found to have fluoride-resistance capacity; however, there hardly any literature discusses the removal of fluoride from water using living bacterial cells. Pseudomonas putida(Figure 1)microbial type culture collection has been found to have fluoride-resistant properties ; however, detailed studies on acclimatization, growth under stress, and fluoride removal under bulk or immobilized phases are not discussed. In the present study, the growth of Pseudomonas putidaMTCC 8104 in N.B. media, its acclimatization in a fluoride environment and under substrate stress, and removal of fluoride in the bulk phase has been conducted [6].

Figure 1: Pseudomonas putida.

Microorganisms

The bacteria used for the study is Pseudomonas putida obtained from our laboratory.

Preparation of Synthetic Solution

1. A synthetic solution was prepared of 20 mg/L fluoride concentration was prepared.
2. Initial pH of the solution was maintained at 7.1±0.1 by using 0.1 N NaOH and 0.1 N HCL.
3. This solution was steam sterilized to 121±0.5°C for 20 min [7].

Acclimatization

Pseudomonas putida was first grown in Nutrient Broth media in a 250ml conical flask. After 24 hours the Nutrient Broth turns viscous indicating significant bacterial growth in the flask.5mg/L of fluoride concentration was added to the above conical flask. Thereafter, the fluorine was periodically added increments in a series of 250ml flasks until the fluoride concentration in the growth media reached 200g/L.

Growth Study

The growth pattern of Pseudomonas putida in Nutrient Broth[Table 1]has been studied in the absence of fluoride.

Growth Media

Table 1: Composition of nutrient broth media.

Standardization of Physical Parameters

Effect of pH

pH considered to be one of the crucial parameters which decide the survival and degradative activity.In the present study,50ml of Nutrient Broth has been taken in 8 conical flask and all were supplemented with

20mg/L of fluoride concentration.The Nutrient Broth was adjusted to different pH using NaOH and HCl.The culture flasks were then inoculated with loopful of bacteria.These were incubated at 30ºC for 24 hours at

125rpm.After 24 hours of incubation, the growth of Pseudomonas putidawas measured at 600nm.

Effect of Temperature

Temperature helps in understanding the ability of bacteria survival at high and low temperature in the present study,50ml of Nutrient Broth has been taken in 6 conical flasks and all were supplemented with 20mg/L of fluoride concentration. The Nutrient Broth was adjusted to optimal pH. The culture flasks were then inoculated with loopfull of bacteria. .These were incubated at different temperatures for 24 hours at 125rpm.After 24 hours of incubation, the growth of Pseudomonas putidawas measured at 600nm[8].

Effect of Contact Time

In the present study,50ml of Nutrient Broth has been taken in 10 conical flasks and all were supplemented with 20mg/L of fluoride concentration. The Nutrient Broth was adjusted to optimal pH of 7. The culture flasks were then inoculated with loopfull of bacteria. .These were incubated at optimal temperature of 37ºC for different contact time at

125rpm. The growth of Pseudomonas putidawas measured at 600nm [9,10].

Effect of Initial Concentration

In the present study,50ml of Nutrient Broth has been taken in 5 conical flasks and all were supplemented with different concentrations of fluoride. The Nutrient Broth was adjusted to optimal pH. The culture flasks were then inoculated with loopfull of bacteria. These were incubated at optimal temperature for 24 hours at 125rpm. The growth of Pseudomonas putidawas measured at 600nm.

Growth Study of Bacteria

The growth pattern of the bacterial strain in the nutrient broth media has been studied at 600 nm in absence of fluoride. From (Figure 2) it is evident that the initial lagperiod is observed up to 2 h of incubation, and a stationary phaseis exhibited after around 18 h of incubation. The highest growthrate is obtained at around 18 h of the incubation time. The bacteriaconsume only organic carbon present in the nutrient broth media. After thelog phase of their growth, the organic carbon is exhausted, as aresult, the number of cells in the media decreases. The lag phaseis supposed to be due to the physiological adaptation of the microbial cell to the culture conditions. This may involve a time requirementfor induction of specific messenger RNA (mRNA) andprotein synthesis to meet new culture requirements. The bacterial culture reaches a stationary phase primarilybecause of exhaustion of the carbon and energy source. When a carbon source is used up, it does not necessarily mean that all growth stops. This is because dying cells can lyse and provide a source of nutrients. Growth based on dead cells is called an endogenousmetabolism, which occurs throughout the growth cycle, but can be best observed during the stationary phase. Another cause of the stationary phase that may be observed is that waste products build up to a point where they are toxic to cells and begin to inhibitcell growth. In the present case, all the mentioned reason may be applicable[11,12].

Figure 2: Growth study of bacteria Pseudomonas putida in nutrient broth media.

Standardization of Physical Parameters

Experiments have been conducted in an incubator/shaker to selectthe pH, temperature, contact time and initial fluoride concentration for theoptimum removal of fluoride from synthetic wastewater.

Effect of pH

(Figure 3) indicates that the percentage removal of fluoride increases with increases in pH up to 7, after which it starts decreasing. The bacteria’s growth is highly sensitive to pH. Therefore, the percentage removal of fluoride is maximum at pH 7. With further increases in pH, the growth of the bacterial cells reduces, which reduces the percentage of fluoride removed [13,14]. 

The effect of pH on the percentage removal of fluoride species byBioaccumulation is shown in Figure 3.

Figure 3: Effect of pH on fluoride removal byPseudomonas putida.

Effect of Temperature

(Figure 4)represents the influence of temperature on fluoride removal. As per literature review, 37 C is considered to be the optimum temperature for bacterial growth. There was a notable change in the amount of fluoride removed with varying temperature, wherein, the highest removal percentage of 61% was observed when the temperature was 37 C. However least removal was observed at 15 and 42 C temperature respectively which can be due to the lower survival capability of the bacterium at minimum and maximum temperature. When the temperature was further increased to 42 C, the removal decreased from 61% to 42%. Further increase in temperature might have also led to disorientation in the bacterial structure and also associated changes in the ion-binding sites. The optimum growth temperature of Pseudomonas putida is reported to be 37 C [15]. 

The effect of Temperature on the percentage removal of fluoride species byBioaccumulation is shown in Figure 4

Figure 4:Effect of temperature on fluoride removal in bulk phase by Pseudomonas putida.

Effect of Contact Time

The effect of contact time on the percentage removal of fluoridespecies by bioaccumulation is shown in (Figure 5)

The effect of Contact Time on the percentage removal of fluoride species byBioaccumulation is shown in Figure 5.

Figure 5:Effect of contact time on fluoride removal in the bulk phase by Pseudomonas putida.

From (Figure5), it is evident that the percentage removal of fluorideincreases with contact time. Around 50.8% fluoride removal is obtainedin a short contact time, i.e., only 3 h and the maximum percentage removal of fluoride achieved by Pseudomonas putidais 92.8% at 25 hours contact time. After around 36 h ofcontact time, further increases in percentage removal of fluoride arenegligible.[16]From these observations, it seems that equilibrium is reached with a contact time of around 25 hours. Fluoride’s toxicity doesnot impose a great effect on culture because a fluoride-acclimatizedculture was used in the present study. After overcoming the inhabitationperiod, the bacteria starts rapid accumulation of fluoride at the initial stage of contact time (∼3 h). Due to the increase in contacttime, the percentage removal is increased up to the equilibriumconditions. After this time period, no further fluoride removal is found due to the arrival of the stationary phase.[17]

Effect of Initial Concentration

(Figure 6)shows the effects of initial fluoride concentration on theremoval of fluoride. 

The effect of Initial Concentration on the percentage removal of fluoride species byBioaccumulation is shown in Figure 6

Figure 6: Effect of initial fluoride concentration on fluoride removal by Pseudomonas putida.

(Figure 6), it can be seen that around 93.4%fluoride removal is achieved for the initial concentration, and aslower as 1 mg/L fluoride and 91.7% fluoride removal is achieved for an initial concentration as high as 20 mg/L after

25 hours of agitation.[18,19].The microbial growth is highly dependent on the initial fluoride concentration. Decrease in the fluoride accumulation rate with increases in initial fluoride concentration in the medium may be due to the toxicity of fluoride to Pseudomonas putidaat higher fluoride concentrations. The removal efficiency is found as 93.7, 92.2, 91.9, and 91.7% for fluoride concentrations of 5, 10, 15, and 20 mg/L, respectively [20].

Optimum conditions for the removal of fluoride from the synthetic water solution observed were pH of 7, temperature at 37ºC, contact period of 25 hours and initial concentration of 5 mg/L.Pseudomonas putidahas a good potential for fluoride removal of up to 5 mg/L.