Introduction

Heavy metals removal by microorganisms is of increasing interest and generally there is an interaction between bacteria and the present metals where bacteria have a role in conversion and transformation of metal ions in different polluted environments (Chang et al., 1993). Moreover, the metal-resistant bacteria from polluted environments are used as indicators of toxicity to different forms of life (Doelman et al., 1994, Hiroki, 1994).  Heavy metal resistant mechanisms of baccteria may due to detoxifying of the polluted environments (Rohit and Sheela, 1994), the genetic transfer of bacteria (De Rore et al., 1994; Goblenz et al., 1994, Guzzo et al., 1994), prevent these metals mobility and bioavailability to the edible plants (Holm et al., 1995).  Many possibilities are found to use these bacteria for metal removal through complex formation of metal ions and different organic acids including citric acid. Biodegradation of the complex of citric acid and metal is depending on the used microbes and nature of the metal (Brynhildsen and Rosswall, 1989). Gram-negative bacterium, Pseudomonas aeruginosa and Gram-positive Bacillus thuringiensis  are resistant to many heavy metals  due to intra- and extra-cellular substance productions (Lima e Silva et al., 1998).

In soil or waste water environments, Cu ⁺⁺, Cr ⁺⁺⁺, Cd ⁺⁺, and Co ⁺⁺ were found and these metals affect bacterial biomass, growth, cell dry weight,   pigment production, ammonium assimilation and protein synthesis. The objectives of this study were to determine the effect of some heavy metals on growth and metabolism of a bacterial isolate belonging to filamentous bacteria.

Material and Methods

The used bacterial isolates

Streptpmyces coeliclor SM1 and S. anulatus SM21 were isolated from shrimp shells on chitin agar medium; containing chitin as sole carbon and nitrogen sources. They were identified according to physiological, morphological, and biochemical characteristics, in addition to 16S rDNA analysis (Aly et al., 2011b). They were kindly obtained from Prof. M M Aly, Biology Department, Faculty of Science, KAU.

Bacterial growth

Bacteria were cultured in starch nitrate broth at a temperature of 30°C and 120 rpm for 7days and in 250 ml flasks containing 50 ml of Starch Nitrate broth (SNB) medium composed of (g/l):  Soluble starch, 10; K₂HPO₄, 2; KNO₃, 2; MgSO₄·7H₂O, 0.5; Ca (CO₃)₂, 2 and one ml of trace element as described by Shirling and Gottlieb (1966).  Each flask was inoculated by 2 ml of the preculture (4×10⁶ cfu/ml) which prepared in SNB medium, for 4 days at 30°C and 120 rpm. At the end of the growth period, culture filtrate was centrifuged for 10 min at 10000 rpm and cell pellet was used for cell dry weight (µg/ml), nitrogen (µg/100 ml) and protein (µg/100 ml) determination.

The two bacterial isolates were grown in different concentrations of the tested heavy metals (Cd:  0.5-1.5 mM, Cr : 0.5-1.5 mM, Co: 0.1-0.5 mM and Cu: 0.5-1.5 mM).  The MIC for each metal was determined as the lowest heavy metal concentration that permit the lowest bacterial growth (the lowest optical densities at 550 nm, OD_{550 nm}) or that  cause ≥ 95% count reduction in cfu/ml (Vela-Cano et al., 2014).

Streptpmyces coelicolor SM1 was grown in SNB medium containing lower concentration of the detected MICs of the different studied heavy metals (1mM for Cd, Cr and Cu but 0.3 mM for Co) and growth, protein, nitrogen content and  % of heavy metal removal were determined after 1, 3, 5 and 7 days as described below. Control flasks without any heavy metal addition was used as control.

The used heavy metals 

Heavy metal stock solutions were prepared by dissolving Cd(NO₃)₂.4H₂O, CoSO₄.7H₂O.  CrSO₄.8H₂O and CuSO₄.5H₂O in distilled water and all the prepared solutions were filter sterilized using 0.45µm blue filter to prevent bacterial growth and all solutions were persevered until used for maximum one month at 4°C.

Protein contents

To solubilize cellular protein, the collected cells were washed, dried and incubated in 5 ml of 1N NaOH for 10 min at 90°C (Jackson et al., 1989). Using the method of Lowry et al. (1951), protein content (µg/100 ml) was determined using bovine serum albumin as protein standard.  All experiments were replicated three times and mean value ± Sd was calculated and compared.

Table 1: Effect of different concentrations of Cd⁺⁺ and Cr⁺⁺⁺  on bacterial growth (dry weight, µg/ml) after growing in starch nitrate broth medium  for 7 days at 30ᵒ C and 120 rpm.

*: significant result compared to control at p ≤ 0.05, S: Streptomyces

Pigment production

Pigment production was determined using naked eye as +++ high pigment production, ++ moderate production, + low pigment production and – no pigment production.

Measurement for dry cell weight (µg/ml)

After bacterial growth, the cells were collected after centrifugation for 10 min at 10000 rpm and the cell pellets were washed and oven dried at 60ºC for 24 hours or until constant weight (Aly et al., 2011a). The experiment repeated three times and the calculated dry cell weights were expressed as µg/ml.

Nitrogen content of the cells (µg/100 ml)

Nitrogen content of the cells was measured using Kjeldahl method (Hawk et al., 1947). The dried cells were digested using concentrated H₂SO₄ in the presence of 1 g of a mixture of CuSO₄.H₂O and K₂SO₄ as a catalyst. Distillation was carried out using NaOH and the evolved NH₃ is absorbed in standard HCl. Using Methyl Red as an indicator, the excess HCl was titrated against standard NaOH  (AOAC, 1990).

Table 2: Effect of different concentrations of  Co⁺⁺ and Cu⁺⁺ on bacterial growth after growth (dry weight, µg/ml) after growing in starch nitrate broth medium  for 7 days at 30ᵒ C and 120 rpm.

*: significant results compared to control at p ≤ 0.05, S: Streptomyces

Metal content

Heavy metal contents (Cd⁺⁺, Co⁺⁺, Cr⁺⁺⁺ and Cu⁺⁺) of the dried and washed bacterial cell sample was determined after digestion using a mixture of HNO₃/H₂SO₄/HClO₄ (2/2/0.5, v/v/v), followed by atomic absorption spectrometry  (Perkin Elmer 2380)   as described in El-Sawi et al. (1994).

Statistical analysis

The obtained data were subjected to Statistical analysis using SPSS program version 16 and the data were represented by means +SD. All the data were compared to control and the difference considered significant at p ≤ 0.05.

Table 3: Growth, pigment and protein production of Streptomyces coelicolor SM1 under normal condition (Control) for 7 days at 30ᵒC, pH7 and 120 rpm

– : No pigment, +: Low pigment production, +++: High pigment production

Results and Discussion

Actually, the existing heavy metals in polluted environments are of great interest and couse serious problems.  The undesirable elements, Cadmium, Chromium, Cobalt and Copper strongly inhibited the bacterial growth of Streptomyces coeliclor SM1 and S. anulatus SM21 (Table 1 and 2).  Streptpmyces coeliclor SM1 was more resistant to Cd ⁺⁺, Cr ⁺⁺⁺, Co ⁺⁺ and Cu ⁺⁺ compared to S. anulatus SM21 (Table 1 and 2) and the inhibition depends on the nature of the metal used and its concentration, and the bacterial isolate examined. Similarly, Lima e Silva et al. (2012) recorded that cell resistant to heavy metals was affected by type of cell, time of exposure and metal type and concentration. Resistant of bacteria to heavy metals may due to biofilms formation (Xu et al., 1998), secretion of detoxifying enzymes or  adsorption of metals by living or dead cells (Hassett et al, 1999).  In control medium, growth, pigment production, N content and protein generally increased by time up to 7 days (Table 3) while they were decreased significantly by the presence of the tested heavy metal. The minimal inhibitory concentration (MIC) of each element was calculated as described by Hassen et al., (1998).  Lower concentrations  of the calculated MIC of each metal was used to determine their effect for 7 days on S. colicolor SM1 growth, pigment production, nitrogen content, proteins and heavy metal removal compared to control (Table 4, 5, 6 and 7). At concentrations below the MIC, Cadmium, Cobalt and Cupper inhibited pigment production by the selected Streptomyces compared to control while the effect of Chromium ions on pigment was not detected. Pigment production by Pseudomonas aeruginosa was affected by the presence of heavy metals (Lima e Silva et al., 2012).

Table 4: Growth,  pigment and  protein production  and  heavy metal removal of  Streptomyces coelicolor SM1 under the presence of 1 mM of Cadmium for 7 days at 30ᵒC, pH7 and 120 rpm in starch nitrate broth. 

–  : No pigment, +: Low pigment production,  ND: Not detected

Table 5: Growth, pigment and  protein production  and  heavy metal removal of  Streptomyces coelicolor SM1 under the presence of 1 mM of Cromium for 7 days at 30ᵒC, pH7 and 120 rpm in starch nitrate broth.    

-: No pigment, +: Low pigment production, ++: moderate pigment production, ND: Not detected

Table 6: Growth,  pigment and  protein production  and  heavy metal removal of  Streptomyces coelicolor SM1 under the presence of 0.3 mM of Cobalt for 7 days at 30ᵒC, pH7 and 120 rpm in starch nitrate broth

– : No pigment, +: Low pigment production,   ND: Not detected

Table 7: Growth, pigment and  protein production  and  heavy metal removal of  Streptomyces coelicolor SM1 under the presence of 1 mM of Copper for 7 days at 30ᵒC, pH7 and 120 rpm in starch nitrate broth.     

-: No pigment, +: Low pigment production,   ND: Not detected

Heavy metals decreased the organic matter formation and accumulation (Vela-Cano et al., 2014) in bacteria which played an important role in waste water and soil cleaning from heavy metals due to bacterial trapping and sequestration. Kathiravan et al., (2010) found that growth, morphology and metabolism of microorganisms present in soil and biological waste water treatment were affected by metals and increasing metal concentration increased lag time and decreased or inhibited growth rate.  Decreasing bacterial growth may be due to inhibition of biodegradation processes of organic compound (Sandrin and Maier, 2003).  In our study, all the tested metals decreased cell nitrogen and protein content.  As well known,  bacteria have the ability to  produce acids, gases, and intermediate products, toxins, agglutinins, and many other protein complexes  during metabolisms and all are affected by the presence of heavy metals (Lima e Silva et al., 2012, Mustapha and Halimoon, 2015).   Metal ions may act as cofactor for many enzymes and are essential for normal bacterial cells  while their excess undergo a Fenton reaction which produced  reactive oxygen species (ROS)  and Thiols,  leading to damage  of the  living cells (Nies,. 1999, Lemire et al., 2013) or  cell toxicity  (Workentine et al., 2008). The harmful effect of heavy metal may due to inhibition of DNA transcription or release intracellular Fe ions into the cytoplasm leading to ROS formation (Xu et al., 2013). In addition, Cr ions inhibit uptake  of sulphate  which is essential element  (Nies and Silver,1995, Holland et al., 2010) while lipid peroxidation was noticed by copper  and cadmium uptake ( Hong  et al., 2012)  Copper ions cause cell lysis due to destruction of extracellular DNA (Dibrov et al., 2002; Warnes et al., 2012) and cations can be pumped out of the cell  (Harrison et al., 2004).  It was found that removal of heavy metal increased by time. After 7 days, Cadmium (38%) and chromium (39%) were the most adsorbed elements by Streptpmyces coeliclor SM1 followed by Cobalt (29%) and Copper (25%). Pseudomonas earuginosa removed Cr ions up to 35-55% and lower removal activity was recorded by Bacillus thuringiesis (Hassen et al., 1998).  Ozturk (2016) reported that S. coelicolor A3(2) can tolerate higher concentrations of heavy metals due to the property of vancomycin which act as a zinc chelator and Streptomyces isolates were heavy metal resistant and had heavy metal binding capacity (Schmidt et al., 2009, Alvarez et al., 2013, Daboor et al., 2014). In conclusion, S. coeliclor SM1can be used significantly to remove Cadmium, Chromium, Cobalt and Copper from contaminated areas.

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