Abdul Latif Ibrahim

Bioremediation of Cyanide by Optimized Resting Cells of Rhodococcus Strains Isolated from Peninsular Malaysia

Five strains of locally isolated Rhodococcus species from various sources in Peninsular Malaysia were evaluated for their cyanide bioremediation prospective. The partially characterized Rhodococcus UKMP-5M which was used as reference strain failed to propagate exclusively in potassium cyanide (KCN). Therefore, the potential use of resting cells grown in nutrient broth without cyanide was tested to detoxify KCN at various concentrations ranging from 3 mM to 15 mM. The physical characteristics for the cultivation of Rhodococcus UKMP-5M namely temperature, pH and percentage of inoculum were optimized in order to be used as source for cyanide treatment. It was found that the maximum biomass was generated amounting to 8.3222 g/L when the incubation temperature was set to 30 °C at pH 6.6 with an initial inoculum loading of 2 % (v/v). Whole cells with biomass amounting to 1 g/L of the strain were able to rapidly transform almost 50 % of 12 mM cyanide over a period of 10 hours. The biotransformation was carried out at 30 °C, pH 7 and with an agitation at 160 rpm in the absence of added organic and inorganic substances. In addition, by employing the identical amount of biomass and other experimental conditions, the different strains of locally isolated Rhodococcus species were compared for their ability to metabolize cyanide. It was revealed that Rhodococcus UKMP-5M had the highest percentage of cyanide removal efficiency followed by Rhodococcus zopfii, Rhodococcus sp1, Rhodococcus NAM81 and Rhodococcus sp2 which corresponded to 47.78 %, 29.17 %, 23.61 %, 18.33% and 11.67 % respectively.

Biodegradation of cyanide by Rhodococcus UKMP-5M

A new bacterial strain, Rhodococcus UKMP-5M isolated from petroleum-contaminated soils demonstrated promising potential to biodegrade cyanide to non-toxic end-products. Ammonia and formate were found as final products during growth of the isolate with KCN as the sole nitrogen source. Formamide was not detected as one of the end-products suggesting that the biodegradation of cyanide by Rhodococcus UKMP-5M may have proceeded via a hydrolytic pathway involving the bacterial enzyme cyanidase. No growth of the bacterium was observed when KCN was supplied as the sole source of carbon and nitrogen even though marginal reduction in the concentration of cyanide was recorded, indicating the toxic effect of cyanide even in cyanide-degrading microorganisms. The cyanide biodegradation ability of Rhodococcus UKMP-5M was greatly affected by the presence of organic nutrients in the medium. Medium containing glucose and yeast extract promoted the highest growth rate of the bacterium which simultaneously assisted complete biodegradation of 0.1 mM KCN within 24 hours of incubation. It was found that growth and cyanide biodegradation occurred optimally at 30°C and pH 6.3 with glucose as the preferred carbon source. Acetonitrile was used as an inducer to enhance cyanide biodegradation since the enzymes nitrile hydratase and/or nitrilase have similarity at both the amino acid and structural levels to that of cyanidase. The findings from this study should be of great interest from an environmental and health point of views since the optimum conditions discovered in the present study bear a close resemblance to the actual scenario of cyanide wastewater treatment facilities.

Cyanide degradation by immobilized cells of Rhodococcus UKMP-5M

Anthropogenic sources contribute to the bulk presence of cyanide, which causes substantial health and environmental concerns. A petroleum-contaminated soil isolate, Rhodococcus UKMP-5M has been verified to efficiently degrade high concentration of cyanide in the form of KCN in our previous study. In order to enhance the cyanide-degrading ability of this bacterium, different encapsulation matrices were screened to immobilize cells of Rhodococcus UKMP-5M for degradation of cyanide. It was revealed that the biocatalyst activity and bead mechanical strength improved significantly when calcium alginate encapsulation technique was employed as compared to free cells. The results also indicated that the immobilized cell system could tolerate a higher level of KCN concentration and were able to support a higher biomass density. In addition, the embedded cells retained almost 96% of their initial cyanide removal efficiency during the first five batches and the entrapped cell system maintained 64% of its initial activity after eight successive batches. The encapsulated beads could be easily recovered from the production medium and reused for up to five batches without significant losses of cyanide-degrading ability, which proved to be advantageous from an economic point of view. From this study, it could be inferred that the novel Rhodococcus UKMP-5M strain demonstrated high cyanide-degrading ability and the optimized calcium alginate immobilization technique provided a promising alternative for practical application of large scale remediation of cyanide-bearing wastewaters.

Enzymatic cyanide degradation by cell-free extract of Rhodococcus UKMP−5M

The cell-free extract of locally isolated Rhodococcus UKMP-5M strain was used as an alternative to develop greener and cost effective cyanide removal technology. The present study aims to assess the viability of the cell-free extract to detoxify high concentrations of cyanide which is measured through the monitoring of protein concentration and specific cyanide-degrading activity. When cyanide-grown cells were subjected to grinding in liquid nitrogen which is relatively an inexpressive and fast cell disruption method, highest cyanide-degrading activity of 0.63 mM min−1 mg−1 protein was obtained in comparison to enzymatic lysis and agitation with fine glass beads. The cell-free extracts managed to degrade 80% of 20 mM KCN within 80 min and the rate of cyanide consumption increased linearly as the concentration of protein was raised. In both cases, the addition of co-factor was not required which proved to be advantageous economically. The successful formation of ammonia and formate as endproducts indicated that the degradation of cyanide by Rhodococcus UKMP−5M proceeded via the activity of cyanidase and the resulting non-toxic products are safe for disposal into the environment. Further verification with SDS-PAGE revealed that the molecular weight of the active enzyme was estimated to be 38 kDa, which is consistent with previously reported cyanidases. Thus, the utilization of cell-free extracts as an alternative to live microbial in cyanide degradation offers numerous advantageous such as the potential to tolerate and degrade higher concentration of cyanide and total reduction in the overall cost of operation since the requirement for nutrient support is irrelevant.

Biodetoxification of cyanide-containing industrial wastewaters by Rhodococcus UKMP-5M

The utilization of cyanide in many industrial processes despite its toxicity generated voluminous effluents, which necessitated treatment prior to discharge into the environment. In the present study, Rhodococcus UKMP-5M was solely employed as biological tool to detoxify three different cyanide-containing industrial wastewaters with different characteristics since initial attempt to employ mixed culture of Rhodococcus strains was unsuccessful due to synergistic interaction which yielded only 19% biodetoxification of 12 mM cyanide. The bacterium was able to achieve 97% cyanide removal efficiency in Wastewater A (pH 8.4) after 19 days of incubation. However, the growing cells needed the supplementation of nutrients, which was uneconomical besides requiring extensive time for complete detoxification. In contrast, the employment of immobilized beads of Rhodococcus UKMP-5M, which required a relatively straightforward method, were cost-effective in detoxifying almost 100% of 15 mM cyanide in Wastewater C (pH 9.5) within a short period of 3 days of incubation. This discovery is highly significant since most wastewaters pHs originating from various industrial applications involving cyanide often range from 6 to 9.5 and this strain is therefore seemingly effective for biodetoxification of cyanide-containing wastewaters with high pH values. In addition, it is interesting to note that the presence of heavy metals in the wastewaters did not inhibit the cyanide-degrading activity of the bacterium. The findings from this study provided essential information to develop a pilot plant on a technical scale for the biotreatment of industrial effluents bearing cyanide, which should be of great interest from an environmental and economic point of views.

Rhodococcus UKMP-5M, an endogenous lipase producing actinomycete from Peninsular Malaysia

Escalation in food industries unctuous wastes has led to serious anthropogenic problems to the environment. Parallel to “green strategy”, growing awareness in biological treatment emphasizes efficacy of enzymatic technology for bioremediation. Pertinently, researchers are in search for new lipase-lipid interaction for improved outcome. Rhodococcus species have documented inadequate evidences on lipase enzyme production. Consequent assessments on Rhodococcus isolates from Peninsular Malaysia have identified twelve promising strains as lipase producer. Interestingly, apart from usual lipolytic behaviour, Rhodococcus sp. exhibited significant level of lipase endogenously, while cryogenic grinding method effectively ruptured the cell. An isolate from petroleum-contaminated site, namely Rhodococcus UKMP-5M, projected the highest level of lipase specificity and has further been optimized. It was found out that the best specificity was apparent in acidic condition (pH 5) with 6% inoculum at 30°C for 72 hours of incubation. Due to high level of mycolic cell-surfactant developed in triacylglycerol supplements, cell lysis was employed with Triton X-100 detergent solubilisation. As a result, oil blend composed of various carbon-chain length fatty acids (composite 2) induces enzyme production extensively. Remarkably, R. UKMP-5M found to cater enzyme production without aid of inducer by nature, but additional carbon source like glucose represses lipase production. Further ability for biological treatment was revealed when the optimized R. UKMP-5M whole cell degraded waste cooking oil significantly by solubilizing fatty acids and commencing conversion into biomass. These qualities resemble practical new lipid-lipase biological lipid rich on-site treatment.

Enhanced cyanide biodegradation by immobilized crude extract of Rhodococcus UKMP-5M

ABSTRACT The capability of the crude extract of Rhodococcus UKMP-5M was enhanced by adopting the technology of immobilization. Among the matrices screened to encapsulate the crude extract, gellan gum emerged as the most suitable immobilization material, exceeding the activity of cyanide-degrading enzyme by 61% and 361% in comparison to alginate carrier and non-immobilized crude extract, respectively. Improved bead mechanical strength which supported higher biocatalyst activity by 63% was observed when concentration of gellan gum, concentration of calcium chloride, number of beads and bead size were optimized. The immobilized crude extract demonstrated higher tolerance towards broad range of pH (5–10) and temperature (30°C–40°C), superior cyanide-degrading activity over time and improved storage stability by maintaining 76% of its initial activity after 30 days at 4°C. Furthermore, repeated use of the gellan gum beads up to 20 batches without substantial loss in the catalytic activity was documented in the present study, indicating that the durability of the beads and the stability of the enzyme are both above adequate. Collectively, the findings reported here revealed that the utilization of the encapsulated crude extract of Rhodococcus UKMP-5M can be considered as a novel attempt to develop an environmentally favourable and financially viable method in cyanide biodegradation.

Decolourization and biodegradation of azo dye methyl red by Rhodococcus strain UCC 0016

ABSTRACT In the present study, locally isolated Rhodococcus strains were attempted as biological tools for methyl red removal, a mutagenic azo dye posing threat to the environment if left untreated. Rhodococcus strain UCC 0016 demonstrated superior methyl red-decolourizing activity of 100% after 24 h at static condition in comparison to Rhodococcus strain UCC 0008 which recorded 65% decolourization after 72 h. Optimization of physicochemical parameters at 30°C, pH 7 and supplementing glucose as the carbon source resulted in improved methyl red-decolourizing activity at static condition and reduced the time taken to achieve complete decolourization by 80%. Higher concentration of methyl red (5 g/L) was able to be decolourized completely within 10 h by adopting the technology of immobilization. The encapsulated cells of Rhodococcus strain UCC 0016 demonstrated higher substrate affinity (Km = 0.6995 g/L) and an accelerated rate of disappearance of methyl red (Vmax = 0.3203 g/L/h) compared to the free cells. Furthermore, the gellan gum beads could be reused up to nine batches without substantial loss in the catalytic activity indicating the economic importance of this protocol. Analysis of methyl red degradation products revealed no germination inhibition on Triticum aestivum and Vigna radiata demonstrating complete toxicity removal of the parent dye after biological treatment. The occurrence of new and altered peaks (UV–Vis and FTIR) further supported the notion that the removal of methyl red by Rhodococcus strain UCC 0016 was indeed through biodegradation. Therefore, this strain has a huge potential as a candidate for efficient bioremediation of wastewater containing methyl red. GRAPHICAL ABSTRACT