Effect of Nickel on Growth of some Actinomycetes Isolated from Three Caves in Saudi Arabia View PDF

Although heavy metals are naturally occurring elements that are found throughout the earth’s crust, most environmental contamination and human exposure result from anthropogenic activities such as mining and smelting operations, industrial production and use, and domestic and agricultural use of metals and metal-containing compounds [1,2]. Environmental contamination can also occur through metal corrosion, atmospheric deposition, soil erosion of metal ions and leaching of heavy metals, sediment re-suspension and metal evaporation from water resources to soil and ground water [3]. It has been reported that natural phenomena such as weathering and volcanic eruptions also lead to heavy metal pollution [4-6].

It has been reported that metals such as cobalt (Co), copper (Cu), chromium (Cr), iron (Fe),magnesium (Mg), manganese (Mn), molybdenum (Mo), nickel (Ni), selenium (Se) and zinc (Zn) are essential nutrients that are required for various biochemical and physiological functions. Inadequate supply of these micro-nutrients results in a variety of deficiency diseases or syndromes [7]. The presence of toxic metals in soil can severely inhibit the biodegradation of organic contaminants [8].

Ni is a trace element for bacteria, serving as an essential component of enzymes such as ureases, hydrogenases, CO dehydrogenases, and enzymes in the metabolism of strictly anaerobic bacteria [9].

Heavy metal contamination of soil may pose risks and hazards to humans and the ecosystem through: direct ingestion or contact with contaminated soil, the food chain (soil-plant-human or soil-plant-animal, human), drinking of contaminated ground water, reduction in food quality (safety and marketability) via phytotoxicity, reduction in land usability for agricultural production causing food insecurity, and land tenure problems [10,11].

Soil microorganisms play vital roles in soil fertility and primary production through organic matter decomposition and nutrient cycling [12]. Fungi and bacteria constitute the main components of the soil microbial biomass and serve as very constructive models for studying the harmful effects of metals at the cellular level [13]. However, several studies indicated that accumulation of heavy metals in soils exert toxic effects on soil microorganisms [14]. Nowadays, there are reports stating that soil microorganisms may adapt to the increased, even toxic heavy metal and other xenobiotics’ concentration in soil by developing various mechanisms to resist heavy metal contamination [15]. Also, studies have represented that long-term heavy metal pollution of soils harm microbial activity, especially microbial respiration [16]. The genus Amycolatopsis, proposed by [17], has been classified in the family Pseudonocardiaceae by the application of the polyphasic taxonomic approach to actinomycete systematics [18,19].

Hamedi J, et al. (2015) [20] reported that highly heavy metals resistant actinomycete strains are amongst them Promicromonospora sp. UTMC 2243 was capable of removing cadmium as high as 96.50%. Various studies stated that heavy metals harming microorganism by disrupting their morphology, growth, biochemical activities and cause reduction in their diversity and biomass [21,22].

Generally, toxic metals cause enzyme inactivation, damage cells by acting as antimetabolites or form precipitates or chelates with essential metabolites [23]. Previous research has found that oxidative deterioration of biological macromolecules is primarily due to binding of heavy metals to the DNA and nuclear proteins [24]. Heavy metals, Cd (II), Cr (VI), Cu (II), Pb (II) and Zn (II) have attracted a lots of researchers because they are widely spread in the environment, and some of them are necessary to plants in small amount but turn out to be hazardous in slightly larger amount than the required consistency [25].

There are other reports on the effects of heavy metals on a range of Streptomyces species [26]. Using microbial biomass for the immobilization and remedy of toxic or rare metals from industrial has become a considerable matter [27]. Microorganisms create mechanisms to tolerate heavy metals by either efflux, complexation, to utilize them as terminal electron acceptors in anaerobic respiration or reduction of metal ions [28]. Heavy metals present a great attraction for several elements such as Sulphur, disrupting enzyme function in living cells by forming bonds with this group. Lead, mercury and cadmium ions damage cell transport process by binding with cell membranes [29]. Three main mechanisms for the binding of metals to bacterial cell walls are known:

• Ion exchange reactions with peptidoglycan and teichoic acid,
• Precipitation through nucleation reactions, and
• Complexation with nitrogen and oxygen ligands [30].

Another report stated that many living bacteria decrease or to shifting toxic contaminants into their less toxic forms [31].

Metal exposure may lead to the establishment of tolerant microbial populations, that are often represented by several Gram-positive genera such as Bacillus, Arthrobacter and Corynebacterium or Gram-negatives, e.g. Pseudomonas, Alcaligenes, Ralstoniaor Burkholderia [32]. Numerous studies also identified several species of bacteria as efficient metal accumulating microorganisms. For instance, Bacillus spp. has been reported to have a high potential of metal sequestration and has been used in commercial biosorbent preparation. Iron- and sulfur-oxidizing bacteria, Thiobacillus ferrooxidans and Thiobacillus thiooxidans, respectively, were enriched from contaminated soil and were able to leach >50% of the metals present (As, Cd, Co, Cu, Ni, V, Zn, B, and Be). Strains of T. ferrooxidanswere able to remove all of the Cd, Co, Cu and Ni [33].

Although cave habitats have been shown to be exceptionally nutrient-limited conditions and diverse microbes are still able to thrive [30]. Cave environments are mostly colonized by unicellular/filamentous microorganisms [34]. Recently, several new members of the genera Catellatospora and Nonomuraea were discovered in caves of Mexico and Northern Thailand [35]. The study aimed to isolation of actinomycstes from caves and screen them for resistance to heavy metals.

Site Description

The study was carried out in the district of Al Saman region located 200 km of Riyadh region, between Riyadh and Al Kharj road, Saudi Arabia. The three caves were interesting and named Mossy, Hotel and Reda caves. All studied caves were located at Al saman area. Their locations were as the following, Mossy Cave: 26°27’33.2?N, 47°14’03.3?E, Hotel cave: 26º 28' 14.2 47º 14' 23.8?E?, and Reda cave: 26°27’10.5?N, 47°15’1.9?E. The visitation of these caves was low because these were not opened for visitors. Morphological interpretation revealed that among these, the first two caves (Hotel and Reda caves) have horizontal road and easy to walk inside while the third one, Mossy cave, has deep pit, with 4 m long, and need ropes and a ladder to go inside.

Collection of Samples

From three caves of Saudi Arabia in Al saman region, north east of Riyadh, nine soil samples were collected from different cave parts, entrance, ground, wall and ceiling in clean polyethylene bags, transport to the laboratory and preserved at 4°C until used. Soil samples from cave ground were taken at 5-40 cm depth [36].

Isolation and Purification of Isolates

One gram of every collected soil was suspended in 9.0 ml of distilled water and mixed with vortex for 5 minutes. Then, 0.1 ml of soil solution was spread on starch nitrate agar medium using spread plate technique. Plates were incubated at 30°C for seven days. The colonies which showed morphological difference were selected and purified. containing in g/L: starch, 20.0; agar, 20.0; K₂HPO₄, 1.0; MgSo₄.7H₂O, 0.50; FeSO₄.7H₂O, 0.01; CaCO₃, 3.0; KNO₃, 2.0; NaCl, 0.50 and 1 ml of trace solution (FeSO4. 7H₂O, 1.0; MnCl₂. 4H₂O, 0.1; ZnSO₄.7H₂O, 1.0 and 100 ml dist. H₂O) and pH was adjusted to 7.0±0.2 prior to sterilization. Petri dishes were incubated at 30°C for 5 days. The colonies which showed morphological difference will be selected. The strains purified by spread plate technique, then, transferred to slant and preserved at 4°C. For long period (more than six months), strains were kept in 20 % glycerol and stored at - 80 °C until used [36].

Screening of Nickel Resistant Isolates

Stock solution of Nickel Chloride (NiCl₂) at 5000 mg/l was prepared by adding five grams of copper to 1000 ml of sterile water. Stock solutions were sterilized for 15 minutes at 110°C [37]. Screening of heavy metal resistance was carried out using different concentration of nickel. The concentrations of Nickel Chloride was ranged from 10, 20, 30, 40, 50, 55, 60, 70, 80, 90, 100, 120, 150, 170 to 200 mg/l for. Each tested concentration was added to starch nitrate medium before sterilization. The pH was adjusted with 1 N NaOH and 1 N HCl to pH 7. This was necessary because the addition of the larger amounts of nickel lowered the pH of the medium.

Determination of Minimum Inhibitory Concentration (MIC) of Heavy Metals

Actinobacterial isolates were streaked on starch nitrate agar medium plates containing different concentrations of nickel until the growth become poor or completely inhibited. The incubation was carried out at 30°C for seven days. The isolate NM20 that proved its resistant for high concentrations of nickel was selected for more detail studies [38].

Determination of Minimum Inhibitory Concentration of a Mixture of Copper and Nickel

NM20 were grown on starch nitrate agar medium containing half MIC of nickel 100 mg/l that determined from pervious experiment with gradual increase of copper concentrations ranged from 5, 10,15, 20 to 25 mg/l.

Characterization of the Selected Isolate

Morphological and biochemical characterization of the selected isolate: Among twenty- one isolates, NM20 was showed the highest maximum tolerant value was selected and characterized using morphologically and biochemically. Characteristic of strain NM20 included Gram reaction, indole, methyl red test, catalase test, oxidase test, starch hydrolysis, gelatine production and melanin pigment production. Sensitivity of NM20 to for different antibiotics by standard disc diffusion method were examined on Mueller- Hinton agar [39].

Colony morphology, soluble pigment production and amount of growth after cultivation on starch nitrate, ISP 2, ISP 4, ISP 5, ISP 7 and ISP 9 agars at 30°C for 5 days. To determine the utilization of the selected isolates on different carbon and nitrogen sources, NM20 was grown on ISP-9 medium containing different carbon source such as glucose, sucrose, starch, lactose, dextrose and maltose, and nitrogen sources such as ammonium sulfate, ammonium chloride, sodium nitrate, potassium nitrate, glycine, peptone, yeast extract, vanillin and asparagine, then all plates incubator for 5 days at 30°C.

Molecular identification of the selected isolate: NM20 was grown in liquid starch nitrate broth for 5 days. The pellets were collected by centrifugation at 1000 rpm for 1 minute and washed twice with sterile distilled water. Genomic DNA extraction and 16S rRNA gene PCR amplification were carried out at King Fahad Medical Center, Saudi Arabia. Amplification was performed using 2.5 μl of the selected primers which were forward 27F (5?-AGAGTTTGATCCTGGCTCAG-3?) and reverse 511R (5?-GCGGCTGCTGGCACCTAGTA-3?).

The effect of different concentrations of the dry weight of the selected isolate: Four lower concentrations, less than MIC and MIC, from Nickel was prepared to test their activities on bacterial growth (dry weight). Erlenmeyer flasks (250 ml capacity) each containing 50 ml of starch nitrate broth containing different concentrations of nickel were prepared to detect the effect of nickel on the dry weight of the selected isolate. The tested nickel concentrations were 50, 100, 150, 180 and 200 mg/l.NM20 were grown on starch nitrate agar then three discs of the isolate NM20 were transferred to inoculate every flask containing the prepared medium with the tested concentration of Nickel. After incubation of the flasks for 7 days in a shaker at 120 rpm at 30ºC, the cell pellets were collected after centrifugation for 20 min at 4500 rpm and the cell pellets were washed and oven dried at 60ºC for 24 hr. or until constant weight.

Factors that Influence Removal of Nickel by the Selected Isolate

The effect of different incubation temperature: The optimum temperature for the growth and removal of nickel were determined for the isolate NM20 in 100 ml Erlenmeyer Flasks, containing 30 ml of sterile of Minimal broth containing in g/l: glucose, 5; (NH₄)₂SO₄, 2; K₂HPO₄, 0.5; MgSO₄·7H₂O, 0.2; FeSO4·7H₂O, 0.01 and pH was adjusted to 7.0±0.2 supplemented with half MIC of nickel which was 100 mg/l. Each flask was inoculated with five milliliters of the preculture (4×10⁶ CFU/ml) which were grown for five days at 30ºC.Then, all flasks were incubated in a shaker at 120 rpm at 25, 30, 35, 45°C for five days. The growth was measured by spectrophotometer at 600 nm and the culture filtrates were centrifuged immediately at 4500 rpm for 20 min to separate cell pellets from the supernatant. The collected supernatants were sending to Centre of Excellence in Environment Studies (CEES), Saudi Arabia to determine nickel concentrations.

The effect of different pH values: The experiment that examined the effect of pH on the growth and removal of nickel was determined in Minimal Medium MM broth amended with half MIC of nickel as described before. The pH of the medium was adjusted to pH 3.0, 5.0, 7.0, 9.0 and 11.0 by adding sterilized 1N NaOH or 1N HCl solution. Then, each flask was inoculated with 5 ml of pre-culture (4×10⁶ CFU/ml), five days old, of the isolate NM20. Incubation was carried out for 5 days in a shaker at 120 rpm at the optimum temperature that was determined from the previous experiment. Spectrophotometer was used to measure the growth at 600 nm. Culture filtrate was centrifuged at 4500 rpm for 20 min to separate cell pellets from the supernatant which was supernatant collected to determine nickel concentrations.

The effect of different concentrations of yeast extract: To evaluate the impact of yeast extract on the growth and removal of copper and nickel, Minimal Medium MM broth was prepared and divided to five flasks. Then, different value of yeast extract (0.1, 0.2, 0.3, 0.4, 0.5 g/l) was added to each flask. The five ml (4×10⁶ CFU/ml) of the pre-culture, 5 days age of isolate NM20 was subjected to each flask containing 30 ml of Minimal broth medium that was supplemented with half MIC of nickel(100 mg/l). The pH was adjusted and incubation of flasks for five days at 120 rpm at the optimum temperature was carried out. The growth was measured by spectrophotometer at 600 nm, then the supernatant was collected to determine nickel concentrations.

The effect of incubation time: Experiment to determine the time required for the growth and removal of nickel was performed using 30 ml of Minimal Medium MM broth contained half MIC of nickel, as described before, at optimum temperature, optimum pH and best concentration of yeast extract that showed the best growth of isolates NM20. Inoculation was carried out using 5 ml of pre-culture (4×10⁶ CFU/ml), grown for 5 days age at 30°C for 5 days. All flasks were incubation in a shaker at 120 rpm for different period 3, 5, 7, 9, 11 days. Spectrophotometer was used to measure the growth at 600 nm. The supernatants were collected to determine nickel concentrations.               

Percentage of Copper and Nickel Removal

Total heavy metal in supernatant was measured by Couple Plasma Emission Spectrometers (ICPE–9000) and the result was compared with control to calculate heavy metal degradation capacity (%) as follows [36]:

Utilized heavy metal (mg/l) = Heavy metal added to the Minimal Medium (mg/l) - Heavy metal at the end of culture (mg/l).

Statistical Analysis

All experiments were performed using the statistical Package for Social Science (SPSS for windows, version 16) (SPSS Inc., Chicago, IL, U.S.A) to compare differences in metal removal among tested isolates. The variability degree of the result is expressed as means. The significant of the difference between samples was determined using t-test. All numeric differences in the data was considered significantly different at the probability level P ≤ 0.05 (IBM Corp. Released 2016. IBM SPSS Statistics for Windows, Version 24.0. Armonk, NY: IBM Corp).

Heavy metals represent the most important component of polluted soil, which significantly impacts soil microbial assemblages by their toxic effect. Our aim was to determine the most resistant strains for nickel to use for bioremediation. A total of 21 actinobacteria strains were isolated from three caves located in Saudi Arabia in Al soman region, north east of Riyadh and their morphological characteristics on starch nitrate agar showed in (Table 1). Actinobacterial resistance to nickel was performed in starch nitrate agar media that supplemented with different concentrations of nickel. In general, all isolates were resistant to nickel, but the level of resistance differed between individual isolates (Table 1) with MIC values varying from 30-200 mg/l. NM20 was showed the most resistance to nickel at 200 mg/l (Figure 1).

Table 1: Showed the morphological characteristics on starch nitrate agar of 21 isolates Minimal inhibitory concentration (MIC) of 21 isolates to nickel.

The isolate NM20 was grown on different growth media starch nitrate, ISP-2, ISP- 4, ISP- 5, ISP- 7 and ISP- 9 agar, then the aerial and substrate mycelia were described in addition to the soluble pigment production. The growth ranged from heavy and moderate to poor (Table 2). For carbon and nitrogen sources, we were grown the selected isolates on ISP- 9 containing different carbon and nitrogen sources and incubator at 30°C for 5 days, the result was summarized in Table 3 and Table 4. Sequence analysis of the 16S rRNA gene is a fast and accurate method to identify the phylogenic position of Actinobacteria. A phylogenetic analysis was performed to identify the selected isolated NM 20 using partial sequences of the 16S rRNA. DNA was extracted from isolate NM20. The concentration of extracted DNA and PCR product were estimated using ethidium bromide staining of agarose gels. The molecular identification of NM20 indicated that NM20 had 95% similarity to Streptomyces coeruleorubidus and was identified as Streptomyces sp. NM20 (Figure 2).

Table 2: The selected actinomycetes NM20 on different media after growth for 5 days at 30?.

Table 3: Antibiotic susceptibility and physiological and biochemical tests of the selected isolate NM20.

Table 4: Effect of different carbon and nitrogen sources in ISP-9 medium on growth of the selected isolate NM20.

To detect the effect of different concentration of nickel (Ni) on the dry weight, the selected isolates was grown in starch nitrate broth medium supplement with different concentration of Ni for 7 days at 30°C. The result illustrated that there is reverse relationship between concentration of nickel and dry weight of selected isolate, whereas the increase in concentration of nickel decrease the dry weight of the isolates (Figure 3) (Table 5).

Table 5: Percentage of nickel removal at different growth temperature, pH, and yeast extract and incubation time by the tested isolate NM20.

The temperature is an important parameter for actinobacterial growth and their heavy metals removal, which affects the growth of the isolate NM20 and removal of nickel. In this experiment, we found that best growth was at 25 °C, pH 11, 0.3 g of yeast extract and at 7 days of incubation period by using atomic spectrophotometer (Figure 3 to Figure 7). The high removal of nickel was occurred at 25 °C, pH 9, 0.3 g of yeast extract and at 7 days of incubation. The removal of nickel at different temperature, pH, time and yeast extract that measured by using ICP- 9000 was described.

In the present study, we aimed to perform isolation of action bacteria from three caves in Saudi Arabia and to test nickel resistant of the isolates. Although many challenges that found in caves include, limited availability of organic matter, variable levels of light and humidity, lack of/limited connectivity to the surface, low or high temperatures, the nature of the hydrological connection between the cave, the surface and the groundwater, exposure to human or other animal visitations, air flow and pressure conditions, and the types and concentration of minerals in the matrix of rock surrounding the cavern [30,40], there are many stadies reported isolation actinobacteria from caves belong to Pseudonocardiaceae and Nocardia ceaefamilies [41] many investigations have identified cultivable members of the genus Streptomyces, which are the most prolific antimicrobial producers [42]. Caves have also been shown to harbour microorganisms that display variable enzymatic and antimicrobial activities, which are different from those observed in other extreme environments, and thus are of great interest to researchers [43]. Cave environments are mostly colonized by unicellular/filamentous microorganisms [30,43, and 44].

Similarly, Amasah R, et al. (2012) [45] studied, characterized and isolated various bacterial strains from Ghar Al Hibashi cave which is located 300 km southeast of Makkah, Saudi Arabia.

Several studies have shown that actinomycetes are often isolated from sites with increased concentrations of Ni, either naturally occurring or from anthropogenic activities. Streptomyces spp. has been shown to be dominant members of the microbial community in soils naturally rich in Ni [46]. In this work, we were found that Amycolatopsis orientalis tolerate nickel up to 200mg/l, so our data supports the role of microorganism in the removal of toxic metals from the contaminated soil to protect the environment. The lowest concentration inhibiting the growth of microbes in nickel-contaminated soils was 100 mg/kg for actinomycetes, 100 mg/kg for bacteria [47]. Streptomyces mirabilis P10A-3 also shows the ability to grow on 100 mmol/l NiCl2 with minimal medium agar, but pigment production seems to be inhibited [48]. Also, Van Nostrand JD, et al. (2007) [49] isolated four actinobacterial strains, among them two of the genus Streptomyces, from contaminated riparian sediments. One of the Streptomyces strains was able to grow on 85.2 mmol/l nickel [50]. Three genera belonging to Pseudonocardiaceae family (order Pseudonocardiales) have showed ability to grow in presence of heavy metals, namely Amycolatopsis, Lentzea, and Saccharothrix [20]. Tirry N, et al. (2018) [51] found that Cellulosimicrobium sp. (CP020857.1) tolerate Ni (NiCl₂) up to 500 mg/L and 400 mg/l with Cu SO₄.

Jurado V, et al. (2005) [45] was isolated Agromyces subbeticus sp. nov., from a cave in the Cordoba area of southern Spain. The effect of copper and nickel concentration on bacterial growth were determined by the dry weights. It was found that increasing in the concentration of heavy metals caused decrease the growth of Amycolatopsis orientalis (NM20), so the dry weight of these isolate decreased by the increasing of the heavy metals compared to control. Cabrero A, et al. (1998) [52] 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 [53]. The factors affect growth and the removal process of heavy metals that were studied are temperature, pH, different concentration of yeast extract and incubation time. In order to optimize these conditions for maximum growth and removal of the isolates NM20, the experiments were conducted with 100 mg/l. It was found that maximum removal was obtained. Temperature, which usually enhances biosorptive removal of adsorptive pollutants when increased by increasing surface activity and kinetic energy of the adsorbate, but which may also damage the physical structure of the biosorbent [54]. Our result was that the growth rate was decreased when temperature increase high than 25°C.

Caves are spread in Saudi Arabia and they represent novel habitats for novel actinomycetes. Many actinomycetes were isolated from caves and they were resistant to antibiotics and heavy metals. The most resistant isolate was selected and identified using morphological and molecular methods. Factors affecting growth in the presence of nickel were studied.

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