ISOLATION AND IDENTIFICATION OF SOIL BACTERIA ABLE TO EFFICIENTLY REMOVE COPPER FROM CULTURE MEDIUMS

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1 ISOLATION AND IDENTIFICATION OF SOIL BACTERIA ABLE TO EFFICIENTLY REMOVE COPPER FROM CULTURE MEDIUMS M. CONSTANTIN, C.D. NEGUT 1, C. BARNA 1, C. CÎMPEANU 1, I.I. ARDELEAN 2 1 Horia Hulubei National Institute for Physics and Nuclear Engineering, Bucharest, Romania 2 Department of Microbiology, Institute of Biology Bucharest, Romanian Academy Received May 28, 2015 Copsa Mica is one of the sites with the highest degree of heavy metal pollution in Romania, the whole area being well known on national and international level for the ecological lack of balance due to the non-ferrous smelter plant. Three bacterial strains were isolated from the soil samples derived from the extraction sit. The growth and development of these bacteria in high concentration of toxic metal environments (Pb, Cd, Zn) represent an important research direction, the understanding of those mechanisms involved in bacterial resistance against the toxic effects of heavy metals and radionuclides contributes to the development of new technologies for treating contaminated water. Concurrently, their capacity of absorbing and accumulating the metals present in a liquid medium was analyzed. In this paper we report the isolation, purification and identification of three bacterial strains from metal contaminated soil (Copsa Mica), strains able to grow in high copper concentration and efficiently remove copper from culture medium. The best removal results were obtained with Bacillus megaterium which removes 99.8 % of the copper present in growth media supplemented with 192 mm Cu 2+. Key words: bioremediation, biosorbtion, copper removal bacteria. 1. INTRODUCTION Heavy metal contamination is one of the most important problems nowadays, worldwide this type of pollution being a major threat for the ecosystem as well as for the health of the population. The metallurgical, mining, fertilizer, pesticide and electroplating industries generate and eliminate in the environment residues that contain different toxic metals [1], including copper. The decontamination of polluted sites is realized using two different processes: physicochemical processes that include evaporation, filtration, ionexchange resins, reverse osmosis, chemical precipitation [2] and biotic methods that imply the use of bacteria in decontamination processes. Rom. Journ. Phys., Vol. 61, Nos. 3 4, P , Bucharest, 2016

2 708 M. Constantin et al. 2 The use of physicochemical methods is widely spread but their efficiency is low due to high costs, low recuperation and the secondary toxic compounds that need to be neutralized [3]. A new approach on this problem is the use of viable/non-viable biomass with special emphasis on bacteria as well as fungi and yeasts [4]. The general interest in biological methods is related to certain main advantages: they can take place at the exact location of the contamination; they have a low negative effect on the environment since they do not generate secondary toxic compounds and are much cheaper than the chemical processes [5]. The bacterial surface is very large and has many ligands for specific heavymetals ions, the biomass can be regenerated by the desorption of metal ions using mild acids, hence the bacterial biomass can be reutilized [6]. There are two major biological mechanisms in which the heavy metal ions are introduced in the cytoplasm: the passive uptake and the active uptake. The passive uptake (biosorbtion) is an independent method of the biological metabolic cycle by which the heavy-metal ions are entrapped in the cellular structures and slowly biosorbed onto the cell; this process includes physicochemical interactions between metal ions and different ligands present on the surface of bacteria [4]. The active uptake (bioaccumulation) is the process by which the metal ions are introduced in the cytoplasm through cell metabolic cycle [3, 5, 7]. The bacterial growth in culture medium supplemented with toxic metals occurs on a wide range of concentrations depending on the type of metal studied [8, 9]. The presence of toxic metals can have considerable impact on microbial population from an aquatic habitat, causing reduction of bacterial biomass in chronical pollution cases or a decrease in the metabolic activity and even the death of the entire bacterial population [8]. A better approach for this problem is to use a bacterial consortium capable to grow in heavy metal contamination medium, and have a well-defined metabolism for bioprecipitation processes [10]. Also the search for these types of bacteria should start in heavy polluted environments where a possible strain can have all the necessary capacities for bioprecipitation [11]. The aim of this study is to isolate bacteria or a bacterial consortium, from a chronically metal polluted soil (Copsa Mica) [10], capable to grow in the presence of high copper concentrations and to remove copper from the growing medium in order to develop a practical method for heavy metal decontamination. 2. MATERIALS AND METHODS 2.1. SOIL SAMPLING, PROCESSING AND STRAIN ISOLATION AND PURIFICATIONS Ten grams of soil collected from Copsa Mica (a well-known heavy metal polluted site in Romania) [12] were aseptically sampled into a sterile Falcon tube and transported to the laboratory for analysis.

3 3 Isolation and identification of soil bacteria able to remove copper 709 Using a sterile mortar the sample was homogenized, weighted and distributed in ten Eppendorf sterile microtubes (one gram in each microtube). In order to resuscitate the bacterial species present in soil, all ten aliquots were hydrated with 100 ml sterile peptone water, magnetically homogenized and incubated at 32 o C for 2 hours. After the incubation period from every aliquot tenfold dilutions were done (1 ml initial suspension + 9 ml sterile peptone water). Because a high bacterial contamination was expected, for every aliquot of soil sample six tenfold dilutions were performed. The sixth and the fifth dilution from every aliquot was then entirely filtrated through 0.45 µm (Merck-Millipore ) hydrophilic filter, and the filters were inoculated at the surface of the Tryptic Soy Agar (Merck ) solid culture medium and incubated at 32 o C SELECTION OF BACTERIAL STRAINS ABLE TO GROW IN THE PRESENCE OF COPPER IONS Initially, there were selected 11 bacterial colonies which were able to grow on Casein Soy Broth (Merck ). The isolated colonies grown on filters were further tested for their copper resistance. Thereby, from every type of colony a bacterial suspension with optical density (λ = 600 nm) = 1 was performed. One milliliter from every suspension was inoculated in 100 ml sterile liquid culture medium, Casein Soy Broth (Merck ), supplemented with Cu 2+ in final concentration of mm. Copper was added to culture mediums before sterilization as copper acetate monohydrate (Merck ). The inoculated bottles were incubated for h at 32 o C to identify the copper tolerant bacteria. After 48 h only three isolates were able to grow at this relatively low copper concentration (0.236 mm); these three isolated and purified strains were identified (see below) and used in further experiments QUANTIFICATION OF DOUBLING TIME FOR SELECTED BACTERIA GROWN IN THE PRESENCE OF DIFFERENT COPPER CONCENTRATIONS Every bacterial isolate was inoculated (in replicates) in liquid nutritive medium at a ratio of 1:4 (50 µl inoculums with optical density, OD = 1, in 200 µl nutritive medium). There were performed two types of controls (each was replicated): the negative control, represented by uninoculated copper supplemented culture medium, and the positive control, namely the inoculated culture medium without copper supplement. The growth of the isolated bacteria was measured as increase in OD (λ = 600) in the growing medium supplemented with five incremental concentrations of

4 710 M. Constantin et al. 4 copper: C1 = 0.236, C2 = 0.472, C3 = 0.944, C4 =1.888, C5 = mm Cu 2+. The inoculation was performed in 96 well microplates and the readings were done hourly for 8 h, the last one being achieved after 28 h from the initial inoculation. The measurements were performed in replicates and using these results the doubling time was also calculated for each bacterial isolate COPPER ELIMINATION FROM CULTURE MEDIA BY PURIFIED STRAINS It was also determined the efficiency of copper removal for the three strains by growing them in liquid culture mediums supplemented with copper to reach the following concentrations: 0.100, 0.200, 0.300, and mm Cu 2+. The experiment of measuring copper concentrations from culture mediums lasted 21 days. The readings were performed weekly using the following protocol: 15 ml of inoculated culture medium were filtrated through 0.45 µm hydrophilic filters into sterile glass tube and processed for atomic absorption analysis. The atomic absorption analysis was performed with an AA Avanta (GBC Scientific) spectrometer [13, 14]. 3. RESULTS 3.1. IDENTIFICATION OF THE PURIFIED BACTERIAL STRAINS Preliminary investigations on the morphology and Gram character of the three soil isolates showed that all of them are Gram-positive rods. The biochemical characteristics (BioMérieux API 50 CHB/E) identify them as belonging to genus Bacillus. An alternative method, based on bacterial fatty acids profile MIDI technique, identifies them as Bacillus megaterium, Bacillus subtilis and Bacillus cereus (Table 1). The predominance of iso and anteiso fatty acids having 12 to 17 carbons as well as the high amount of C15:0 iso and anteiso and C17:0 iso and anteiso fatty acids strengthen the identification of this isolate as Bacillus [15, 16]. The high amount of fatty acids is explained by high membrane fluidity of Bacillus genus [15]. The identifications were performed using a gas chromatograph (GC 6890N) coupled with a mass spectrometer (Agilent Technologies). Fatty acids interpretation was performed using an internal software (MIDI Sherlock v6.1) against MIDI fatty acids library RTSBA 6.

5 5 Isolation and identification of soil bacteria able to remove copper 711 Table 1 Three unknown bacterial soil isolates, identified by fatty acids profile as resulted from GC-MS readings, using RTSBA6 library Fatty acid name Fatty acid concentration Unknown 1 Unknown 2 Unknown 3 11:0 iso :0 iso :0 (acid lauric) :0 iso :0 anteiso : :0 iso (12-methyltridecanoic) :0 (acid miristic) :0 iso (13-methyltetradecanoic) :0 anteiso (12-methyltetradecanoic) :1 w5c :1 w7c alcohol :0 3OH/16:1 iso I :0 iso (14-methylpentadecanoic) :1 w11c :1 w6c/16:1 w7c :1 w5c :0 (acid palmitic) :0 2OH :1 iso w10c :1 iso w5c :0 iso 3OH :0 2OH :1 anteiso A :1 anteiso B/iso I :0 iso (15-methyltetradecanoic) :0 anteiso (14-methylhexadecanoic) :0 (acid margaric) :0 iso :1 w9c :1 2OH :0 (acid stearic) :0 iso :0 anteiso Fatty acid identification (base on GS-MS analysis) Bacillus subtilis Bacillus cereus Bacillus megaterium Index similarities compared with RTSBA 6 database

6 712 M. Constantin et al THE GROWTH OF ISOLATED BACTERIAL STRAINS IN ENVIRONMENTS WITH HIGH CONCENTRATIONS OF COPPER 2,0 2,0 1,8 1,8 1,6 1,6 Optical density (600nm) Growth evolution in medium without Cu Bacillus subtilis ( Boltzmann) Bacillus cereus ( Boltzmann) Bacillus megaterium ( Boltzmann) Optical density (600nm) Growth evolution in medium with 36 mm Cu Bacillus subtilis ( Boltzmann) Bacillus cereus ( Boltzmann) Bacillus megaterium ( Boltzmann) ,0 2,4 1,8 2,2 1,6 2,0 1,8 Optical density (600nm) Optical density (600nm) 1,6 Growth evolution in medium with 72 mm Cu Bacillus subtilis ( Boltzmann fit) Bacillus cereus ( Boltzmann fit) Bacillus megaterium ( Boltzmann fit) Growth evolution in medium with 0,944 mm Cu Bacillus subtilis ( Boltzmann) Bacillus cereus ( Boltzmann) Bacillus megaterium ( Boltzmann) ,0 1,8 1,6 Optical density (600nm) Optical density (600 mm) Growth evolution in medium with 1,888 mm Cu Bacillus subtilis ( Boltzmann) Bacillus cereus ( Boltzmann) Bacillus megaterium ( Boltzmann) Growth evolution in medium with 3,776 mm Cu Bacillus subtilis ( Boltzmann) Bacillus cereus ( Boltzmann) Bacillus megaterium ( Boltzmann) Fig. 1 Growth of B. subtillis ( ), B. cereus ( ), B. megaterium ( ) in 5 copper concentrations (2 6) compared with the control (1).

7 7 Isolation and identification of soil bacteria able to remove copper 713 The growth of the three soil isolates in the presence of copper is different for each, thus, B. cereus and B. megaterium grow faster in copper supplemented culture mediums in concentrations ranging from to mm. Their growth can be compared to that of the control (no copper addition) and after about 4 h their initial concentration is doubled. These two species are better adapted for growing in high metal concentrations and they develop a cellular mechanism in order to neutralize cooper s toxic effects. Among these mechanisms, metal complexation and bacterial immobilization are the most common. For B. cereus and B. megaterium the growth started after 3 4 hours from the initial inoculation and after 23 hours the stationary phase occurred. The growth can be described by a Boltzmann sigmoidal function [17]. B. subtilis showed an atypical growth evolution. The growth started with a delay of 5 hours and after 23 h the stationary phase occurred. After 26 hours another growth phase was noticed. This pattern was detected for all concentrations of copper supplemented mediums. The growth of all three Bacillus species in copper supplemented culture medium started after a latency of about 2 hours. Based on these experimental results the doubling time was calculated (Table 2) with the following formula N(t)=N 0 *, where: N 0 initial concentration, N(t) concentration at the time t, t2 doubling time. Table 2 Doubling time of Bacillus megaterium, Bacillus cereus and Bacillus subtilis grown in mediums supplemented with different copper concentrations compared to the positive control (no copper addition); data calculated according to Ref. [18] Positive control Copper concentration (mm) doubling time, t2 (h) Bacillus megaterium Bacillus cereus Bacillus subtilis As shown in Table 2, in this range of concentrations the doubling time of Bacillus megaterium and Bacillus cereus is practically not influenced by the presence of copper in the culture mediums, a phenomenon which deserves further attention. In both cases, at the end of the growth experiments a bacterial deposit was noticed at the bottom of the inoculated bottles, representing bacteria that absorbed copper.

8 714 M. Constantin et al COPPER REMOVAL BY COPPER RESISTANT BACTERIA The interaction with copper of the three isolates is macroscopically visible both in culture liquid and in solidified culture medium, suggesting that the dark aspect of the colonies (results not shown) in the presence of copper is related to copper removal from the growing medium. For all three isolates the copper absorption capacity was further tested at four experimental concentrations: 0.08, 0.192, 0.308, mm Cu 2+. These concentrations and the incubation temperature were selected in agreement with literature [9, 19]. Figure 3 shows the evolution of all four copper concentrations in the presence of B. subtilis, B. cereus, and B. megaterium for 21 days. After 48 hours from the inoculation, the culture medium became turbid thus indicating bacterial growth; daily, the ph of the culture mediums was measured because the solubility of toxic heavy metal increases with the decrease of ph [13, 14]. After 21 days a decrease of the ph was registered, from an initial value of 7.2 to , with an intense decrease in the first 10 days (ph about 5.9). The copper concentration, at all values, in the presence of all three Bacillus strains displayed a rapid decrease in the first two weeks after the inoculation, when the copper concentration acquired the lowest values. After 14 days equilibrium was acquired and the copper concentrations remained constant until the end of the experiment. As shown above, at all concentrations, Bacillus megaterium has the highest copper removal capacity from the growing medium. The capacity of Bacillus megaterium to remove copper from the growing medium, initially having 100% copper concentration, is also presented in Table 3. Table 3 Copper removal by Bacillus megaterium from growing medium supplemented with different copper concentrations Strain Bacillus megaterium Initial copper concentration (mm Cu 2+ ) removed copper after 7 days (%) removed copper after 14 days (%) Final copper concentration (%)

9 9 Isolation and identification of soil bacteria able to remove copper B.subtilis B.cereus B.megaterium 0 B.subtilis B.cereus B.megaterium 7 0,18 Copper concentration (mm) Copper concentration (mm) 0,16 0,14 0,12 0, Readings (days) Readings (days) 0,32 0, B.subtilis B.cereus B.megaterium 0 0,35 Copper concentration (mm) ,18 0,16 0,14 0,12 0, Copper concentration (mm) 0, ,15 0,10 5 B.subtilis B.cereus B.megaterium Readings (days) Readings (days) Fig. 2 The evolution of copper absorption by B. subtillis, B. cereus and B. megaterium, grown in medium supplemented with 0.080, 0.192, 0.308, and mm Cu DISCUSSION In this paper we report the isolation, purification and identification of three bacterial isolates, namely B. subtillis, B. cereus and B. megaterium able to grow in high copper concentration and able to remove copper from culture medium. Each strain has its own profile for copper removal from growing mediums supplemented with different copper concentrations. The largest decrease in copper concentration in the growing medium is always found in the case of B. megaterium, with a removal capacity of 99 % for lower copper loadings (0.080 or mm Cu 2+ ) and of 50% for higher loadings (0.414 mm Cu 2+ ). The results obtained with these three isolates, namely B. subtillis, B. cereus, and B. megaterium, are in agreement with the performance of a strain of Bacillus

10 716 M. Constantin et al. 10 cereus, which removes copper with a maximum efficiency of 70 % for initial concentrations between mg/l, as well as with other results obtained with bacteria belonging to genus Bacillus [19, 20]. The mechanisms involved in processes of copper removal by use of bacteria are categorized as active and passive [8]. In the Gram-positive cell walls, anionic sites that are important in binding metals are associated with the following: free carboxyl groups of the peptidoglycan, hydroxyl groups of sugar in both peptidoglycan and teichoic acids, phosphodiesters in teichoic acids and amide groups in peptides [8]. For example, the relative affinities of the cell wall of Bacillus subtilis for metal ions can be divided into three classes. The greatest binding capacity occurs with Na +, K +, Mg 2+, Fe 3+, and Cu 2+ with lesser amounts of binding by Mn 2+, Zn 2+, Ca 2+, and Ni 2+. Cations that showed either very little or no binding includes Hg 2+, Ag +, Sr 2+, Pb 2+, Li +, Ba 2+, Cd 2+, and Al 3+. With respect to the mechanism(s) involved in copper removal by the bacterial strains used in this experiment, copper removal by growing cells suggests that active mechanisms are involved [5]. However, further research is needed for a deeper understanding of the processes, especially in the following directions. The copper removal capacity also in the presence of dead cells and the possible variations of the valence of copper ions added to the culture mediums with growing cells. Consequently, further improved research and study will lead to the development of an experimental model for future applications, including bioremediation of polluted environments with different toxic metals like radionuclides. 5. CONCLUSIONS This paper shows the results concerning the isolation, purification and identification of three soil bacteria (Bacillus subtillis, Bacillus cereus, and Bacillus megaterium) able to grow in high copper concentration, 0.236, 0.472, 0.944, 1.888, and mm as well as copper removal capacity during bacterial growth in mediums supplemented with copper in the range mm Cu 2+. Out of the three bacterial isolates, B. megaterium showed the best capacity of copper removal: 99 % for lower copper loadings (0.080 or mm), 75% for intermediate loadings (0.308 mm) and 50% for higher loadings (0.414 mm), abilities which turn it into a very promising candidate for further research.

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