Potency of Indigenous Bacteria of Mt. Merapi, Arthrobacter chlorophenolicus for Chromium (VI) Bioremediation

Chromium (VI) in the production process, such as textile, tannery, and electroplating industry, produce hazardous waste when disposed of directly into the aquatic environment. Several chromium pollutions cases, not only in water but also in the aquatic organism, occurred in some regions in Indonesia. Various methods can reduce the Chromium (VI) waste. One of them is the biological method by employing such kinds of bacteria. Arthrobacter chlorophenolicus is a pioneer bacterium of Mt. Merapi, which can survive in the minimum conditions of the bacterial primary nutrients, carbon, and nitrogen. This study aims to investigate the ability of A. chlorophenolicus to remove Cr (VI) at various concentrations. The research was carried out by growing the A. chlorophenolicus into two nutrient media conditions, minimal and rich-nutrient media containing different concentrations of Cr (VI) (5, 10, 20 ppm) for eight days. The results showed that the A. chlorophenolicus were grown on both minimal and rich-nutrient media. The A. chlorophenolicus could reduce for about 80% of 10 and 20 ppm chromium in eight days. Our results indicate that A. chlorophenolicus, the pioneer bacteria of Mt. Merapi, has a grand promise for use in Cr (VI) remediation even under minimum nutrients conditions.


Introduction
Chromium (Cr) is naturally abundant in Earth's mantle and released by weathering process into water or soil. It can also be produced by industrial processes, such as textile, electroplating, tannery industry, and many so on. Chemically, chromium is one heavy metal that exposes a complex element. Marina [1] explained that Cr in water mainly exists in two oxidation states, i.e., Cr (III) and Cr (VI), depending on pH, potential redox value, and other reducing agents. As well as Cr (III) and Cr (IV) present different chemical, toxicological, and epidemiological characteristics [2]. Chromium hexavalent toxicity is known as highly toxic, while Cr (III) is less mobile toxic. Cr (VI) pollution in the environment is attracting attention since it is widespread worldwide, with high levels in water and soil as a result of natural and anthropogenic processes [3,4]. Furthermore, the poisonous Cr (IV) also affects organisms and microorganisms. The risks to human health range from skin irritation to DNA damage and cancer formation, depending on the dose, degree, and duration of exposure [1].
Among environmental problems, heavy metal pollution in developing countries still becomes a considerable challenge to prevent and solve. For supplying basic requirements of domestic and maintaining economic growth, numerous industries are built and operated. Nevertheless, most of them tend to be careless and have minimum treatment for both solid and wastewater. Specifically, chromium is mainly identified in the textile, tannery, and electroplating industries in Indonesian cases. Asmadi [5] explained tannery industry in Semarang city use a colouring agent containing chromium metal. A study by Murniati [6] showed that the production of Batik, an intangible heritage craft awarded by UNESCO, resulted in wastewater containing heavy metals such as chromium. Besides, Agustina et al. [7] reported that the electroplating industry in West Java resulted in wastewater containing chromium hexavalent up to 750 ppm. The improper utilizing and treatment of wastewater containing Cr (VI) is the leading cause of artificial chromium pollution in the environment.
Unfortunately, several chromium pollutions cases, not only in water but also in the aquatic organism, occurred in some regions in Indonesia. In Jember, East Java, an electroplating industry reported directly throwing their wastewater in the water stream without treating accomplishment. Bedadung River around the industry-accepted discharge water resulted in the water river quality over the allowed standard. Chromium concentration reached 0.15 mg/L exceed the maximum permitted standard by the government, which is 0.05 mg/L [8]. Even though chromium concentration in Winongo River Yogyakarta was below the maximum standard, chromium inside Red Tilapia (Oreochromis sp.) edible fish tissue nurtured in the fishing net surpassed the National Agency of Drug and Food Standard. The limitation allowed to be consumed by a human is 2.5 mg/Kg, but in Tilapia, muscles pointed on 10.2265 mg/Kg, 9.8107 mg/Kg, and 9.2245 mg/Kg in Station 1 to 3, respectively [9]. A higher concentration of chromium in fish tissue than in water exposes the heavy metal possessed bioaccumulation characteristic due to high solubility of Cr (VI) as the toxic ion is transferred quickly through the food chain. River and fish contaminated by chromium hexavalent as the source of water and food can be dangerous for human health. Cr (IV) causing liver and kidney damage, internal bleeding, and breathing problems, so that the International Agency for Research on Cancer has classified it as carcinogenic to humans (Group I) [10,11].
Some strategies are applied to treat and remediate toxic Cr (VI) include chemical reduction, precipitation, adsorption, ion exchange, electrocoagulation, or biological reduction [1]. Each method has advantages and disadvantages. By comparing chemical and biological methods for removing Cr (VI), the most suitable strategy is based on a requirement of case, site, and the initial evaluation. According to Majone [12] and Linkov [13], especially for in-situ interventions, which necessitate a complete understanding of contaminated matrix geochemistry, hydrogeology, microbiology, and ecology. Moreover, cost-benefit analysis is compiled frequently and in conjunction with comparison risk assessment.
Based on research history in Yogyakarta, to mitigate chromium pollution, a combination of phytoremediation and adsorption was employed to treat tannery Cr-wastewater [14] by selecting chromium reduction bacteria in the waste of tanning leather industries by ozone method [15] and biosorption using biomass of Fusarium sp. and Aspergillus niger for reducing bearing tannery wastewater [16]. A new approach is to remove Cr (VI) contamination using the bioremediation method that employs indigenous bacteria isolated from the Mt. Merapi volcanic deposit after two years of eruption. The bacteria were identified as pioneer bacteria that can survive in the minimum condition of the primary nutrients, Carbon (C) and Nitrogen (N), namely Arthrobacter chlorophenolicus [17]. Other studies investigated the potency of Arthrobacter sp. to mitigate chromium hexavalent from wastewater, for example, Molokwane [18] using Arthrobacter sp., which was able to reduce Cr (VI) up to 94.3% from the initial concentration of 100 mg/L in 24 hours of incubation. In addition, Dey and Paul [19], using the Arthrobacter sp. SUK 1201 as a remediator showed a 67% reduction in 2 mM Chromate within seven days of incubation. Thus, this study aims to investigate the ability of indigenous bacteria of Mt. Merapi, Arthrobacter chlorophenolicus, to remove Cr (VI) at various concentrations and to observe the effect of incubation time on the ability to reduce Cr (VI) in wastewater. Hence, this study is an early stage of applying that microorganism on bioreactor in the later studies to reduce heavy metals in wastewater and by releasing them to mitigate Cr (VI)-contamination in water resources.

Sample Collection
Bacterial isolates, Arthrobacter chlorophenolicus, were isolated from the volcanic deposit of Mount Merapi, Special Region of Yogyakarta Province, as identified by Lathifah et al. [17]. The source of Cr (VI) utilized in this study is the K2Cr2O7 compound named synthetic wastewater. The synthetic wastewater receipt is referred to from Rcheulishvili [20]. Some various treatments are occupied, including the incubation time and Cr (IV) concentration. The time incubation series are 24, 120, and 168 hours; also, the Cr (IV) concentration is in the range of 5, 10, 20 ppm. Some nutrition, such as NaCl (Sodium Chloride), KH2PO4 (Monopotassium Phosphate), Na2HPO4 (Disodium phosphate), NH4Cl (Ammonium Chloride), MgSO4 (Magnesium Phosphate), CaCl2 (Calcium Chloride), and D-Glucose are added in the medium, and as control of the treatment is no additional nutrition.

Isolate preparation and acclimatization
Arthrobacter chlorophenolicus isolated from the previous research of Lathifah et al., [17] was inoculated into Nutrient Broth media at 30°C for 48 hours in a shaker incubator. The minimum populations of cell numbers are notified by 10 9 CFU/ml or equal to 0.4 A [21]. Furthermore, the isolate was acclimatized in 5 ml of media containing five ppm Cr (VI), ready for the running stage [22].

Running stage
For testing the ability of Arthrobacter chlorophenolicus to remove Cr (VI) was carried out on a laboratory scale by analysing its growth ability in media containing chromium hexavalent in various concentrations (5, 10, 20 ppm) using the batch culture method [23]. In this study, the growth media used were specific mineral media. The composition of the media (in each g/L) is Na2HPO4 (1.264); KH2PO4 (0.326); NH4Cl (1); MgSO4 (0.098); CaCl2 (0.044); and Glucose (0.1). The media was added by K2Cr2O7 solution as a source of Cr 6+ [24]. More detailed procedures are explained as follows: (1) Media preparation Specific mineral media added with K2Cr2O7 reagent were prepared to obtain a specific media solution with a concentration of 5, 10, 20 ppm of Cr (VI). The various K2Cr2O7 solutions were prepared by diluting 20 ppm of K2Cr2O7 solution to get ten ppm and five ppm [24].
(2) Inoculation of Arthrobacter chlorophenolicus in growth media An Arthrobacter chlorophenolicus of 5 ml was prepared in growing media + Cr (VI) at various concentrations on a batch reactor illustrated by Fig. 1. After that, it is incubated with a shaker using an orbital shaker at 30°C for 192 hours or eight days. At the 0, 24, 120, and 192 hours, every solution sample was taken to analyse bacterial growth using an OD spectrophotometer. Then, at 0 and 192 hours, the Total Plate Count (TPC) was taken, followed by pH and temperature measurements. The concentration of media containing Cr (VI) was known initially so that the test was run only for 192 hours or the last day of the running step [20]. As a control, the same media was used without the addition of Arthrobacter chlorophenolicus. Blank samples and treatments are evaluated in duplicate with each set of samples.  The capability of bacteria growth on media testing was observed OD spectrophotometer. Briefly, in the OD spectrophotometer method, as much as 3 mL of liquid media from the growth media, including Cr (VI), was inserted into the cuvette. Then, the absorbance value was measured using a spectrophotometer with a wavelength of 600 nm. Likewise, the colony OD test was calculated at an amount of 10 9 CFU/ml or equal to 0.4A measured by OD spectrophotometer [22]. (

4) Cr (VI) Reducing Analysing
Analysis of Cr (VI) reduction was accomplished by using a UV-Vis spectrophotometer. Test samples at 0; 24; 120; and 192 hours were observed for Cr ions removal with a wavelength of 535.9 nm [20].

Data analysis 2.4.1 Removal efficiency of Cr (VI)
The Chromium removal efficiency was obtained after testing through the UV-Vis spectrophotometer before bacterial cultured and after the running stage. Based on the test, the metal removal efficiency can be calculated according to spectrophotometer analysis. The following is a formula for calculating the efficiency of metal removal [26]: Removal Percentage %= C0-CC0 x 100% (1) With C0 = Initial concentration (mg/L) and C = Later concentration (mg/L) (2)

Graph analysing
Bacterial growth can be determined by using OD Spectrophotometer. The bacterial growth can be recognized by an increase of OD on the tested media. In addition, the metal reduction can be detected by using a spectrophotometer with a concentration ratio between incubation time and the growth of bacteria. The decreased Cr (VI) concentration employing A. chlorophenolicus is estimated by more than 60% removal efficiency percentages.

Results
Growth of Arthrobacter chlorophenolicus (A. chlorophenolicus), indicated by the increasing of OD, as well as hexavalent chromium (as K2Cr2O7) reduction, was monitored at different initial chromium concentrations of 5, 10, 20 ppm both in Nutrient Broth (NB) media and specific mineral media during 192 hours incubation. The A. chlorophenolicus growth and Cr (VI) reduction was evaluated at 0, 24, 120, 192 h. Cr (VI) reduction was found to increase proportionally with the increase in bacterial growth indicated by the increase of OD of the culture. Chromate reduction occurred rapidly in NB (Fig. 2a) and mineral media (Fig. 2b). In NB media, the percentage removal of chromium by A. chlorophenolicus in culture A (5 ppm Cr), culture B (10 ppm Cr), and culture C (20 ppm Cr) was 40%, 30%, 25%, respectively, after 24 hours incubation. With further incubation by 120 hours, about 50% of the Cr (VI) added in each tested culture were reduced. After 192 hours of incubation, the added Cr (VI) was removed  (Fig. 2a). The percentage removal of chromium results in minimal media was not much different from the results of the NB one. The removal of chromium occurred after 24 hours incubation and reached about 50% after 120 hours incubation. With further incubation, 192 hours incubation, the percentage removal of chromium by A. chlorophenolicus in the culture D (5 ppm Cr), culture E (10 ppm Cr), and culture F (20 ppm Cr) was 60%, 74%, 79%, respectively (Fig. 2b). During the growth of A. chlorophenolicus in reducing chromium, each culture's pH and temperature were observed at 0 and 192 hours incubation. The results showed that the pH value at 0 h was recorded for 5-6 and increased to seven after 192 hours incubation in all the tested cultures (Fig. 3a). The temperature also was observed for all the tested cultures at the same time. The results showed that the temperature was constantly at 26-27°C in all the tested cultures (Fig. 3b).

Discussion
Bioremediation of heavy metal pollution is an attractive solution that is cost-effective and environmentally friendly. Several studies have been reported on chromate reduction by Arthrobacter spp., members of actinomycetes genus isolated from soil contaminated with tannery effluent [27], basalt rocks [28], activated sludge [18], and landfarming process soil sample [29]. These studies also reported its application in bioremediation of Cr pollutant in culture scale. Further, the following study by Dey et al. [21] in their recent study [19] reported optimizing cultural conditions for the growth of Arthobacter spp. to evaluate their removal efficacy. However, the study of Arthrobacter spp. isolated from the volcanic area, specifically from volcanic deposit and evaluation of its potential for bioremediation of chromium has not yet been reported before. This study using Arthrobacter isolate from the previous study by Lathifah et al. [17], which is characterized to be able to grow in the minimum conditions of the primary bacterial nutrients, Carbon (C) and Nitrogen (N). Thus, it is a beneficial advantage when the isolate will be applied for bioremediation on a large scale because the addition of C and N sources in large amounts is not needed in maintaining the culture.
In this study, we differentiate the growth of A. chlorophenolicus in rich media, namely Nutrient Broth (Fig. 2a) and minimal nutrient media, namely mineral media (Fig. 2b), which both containing Chromium in various concentrations (5, 10, 20 ppm). The results showed that A. chlorophenolicus grew in the minimal nutrient media, as can be seen, based on the increase of the OD value. When comparing with the growth of the isolate in the NB media, rich nutrient media, the growth was comparable even though the growth in NB media seems relatively higher than in the minimal one. It is expected to occur because the growth rate of bacteria depends on environmental conditions. If the environmental conditions have low nutrition, the general growth will be slower than the growth of bacteria in the nutrient-rich medium [31].
The percentage removal of chromium results indicated that A. chlorophenolicus showed about 80% reduction of 10 and 20 ppm chromium in 8 days ( Fig. 2a and Fig. 2b). The result suggested that the isolate could reduce chromium hexavalent almost completely even under minimal nutrient conditions (Fig. 2b). Meanwhile, in the concentration of 5 ppm chromium, the A. chlorophenolicus showed about 60% reduction in both NB and mineral media. When observing the growth of A. chlorophenolicus in the media containing five ppm chromium, the growth rate is not as rapid as the isolate's growth in the media containing ten ppm and 20 ppm chromium (Fig. 2a). It might indicate that the amount of the bacterial cell in each culture during the incubation is different, resulting in a lower percentage of chromium removal in culture A and culture D. only use the energy. When compared with the previous study of hexavalent chromium remediation by Arthrobacter spp., these results seem nearly identical with the recent study by Dey et al. [19], which resulted in the reduction of 0.5 mM Cr (VI). Thus, the isolate could be promised bacteria for use in the bioremediation of chromium pollution.
In this study, the pH and temperature were observed during the running cultures. The reduction of Cr (VI) by A. chlorophenolicus is quite good when the initial pH of the media is maintained at pH 4 to 8 [21] [32]. The A. chlorophenolicus effectively reduced chromite in a narrow range of pH with an optimum of pH 7 (Fig. 3a), similar to those results of Camargo et al. [32] and Dey et al. [21]. In addition, the parameters temperature plays a vital role in the growth of A. chlorophenolicus, which will grow optimally at a temperature of 20-40°C, characteristic of mesophilic microorganisms [21]. In this study, the temperature of 26-27°C could be maintained in all the tested cultures until the final run of culture at 192 hours incubation (Fig. 3b).
The study by Molokwane et al. [18] using Arthrobacter isolated from dried activated sludge while Dey et al. [12] [19] using Arthrobacter sp. isolated from chromite mining in their study. In other words, that Arthrobacter spp. isolated from such heterotrophic conditions, ordinary Carbon and Nitrogen contents. Meanwhile, our study using Arthrobacter isolated from such oligotrophic conditions, mostly zero Carbon and Nitrogen contents, which imply the ability of the isolates to survive in such harsh conditions. Thus, our finding might have such a positive impact when the isolate is applied for bioremediation on a large scale because adding C and N sources in large amounts is not needed to maintain the culture.

Conclusions
Our results indicate that A. chlorophenolicus, the pioneer bacteria of Mt. Merapi, has a grand promise for use in Cr (VI) remediation even under minimum nutrients conditions. Further studies in optimizing the culture condition to improve the chromium removal efficacy are now in progress.