Morphological characterization of gram-positive and gram-negative bacteria from treated latex processing wastewater

A preliminary morphological screening and isolation of bacterial colony from latex industrial wastewater was carried out. Bacteria colonies from latex processing wastewater were isolated from a local latex processing industry. It was found that 17 bacterial isolates had been purified grown on nutrient agar under 35 oC. The colonies were then purified and morphologically indicated via Gram staining and motility test. After morphological observation, it was identified that out of 17 isolates, 9 isolates were Gram positive and 8 isolates were Gram negative. There are 11 out of 17 colonies were rod-shaped bacterial colonies, while the other 6 colonies were cocci-shaped bacteria. There were 11 colonies of gliding bacteria, three colonies were non-motile bacteria and the other three colonies were flagellated bacteria. This study investigates the potential occurrence of viable growth in treated latex processing wastewater. The bacterial colonies were classified base on their morphological properties shown. This study has classified several genera such as Staphylococcus, Escherichia, Thiobacillus, Arthrobacter and other Genus. The growth curve of 17 isolates studied and the chemical oxygen demand were determined.


Introduction
Malaysia is among the largest rubber producer in the world after Thailand, Indonesia, Vietnam, India and China. In South-east Asia, about to % of produced raw rubber in the world supply comes mainly from Thailand, Malaysia and Indonesia (Andersen et al., , Mohammadi et al., ). Latex industry produces a massive amount of wastewater daily, mainly coming from the manufacturing process. Latex processing wastewater (LPW) contains high level of Chemical Oxygen Demand (COD), Biological Oxygen Demand (BOD), solids, nitrate and heavy metal which affects the water quality. Illegal discharge of untreated wastewater into the rivers and streams cause various environmental pollution issues. Previously, latex processing industry produced tons of latex and liters of effluent per day in average (Mohammadi et al., ). In natural rubber processing plants, a ton of Standard Malaysian Rubber Block produced with . m effluents which containing . kg of BOD (Sharib and Halog, ). Latex processing effluent generally included wastewater, small amounts of uncoagulated latex, carbohydrates, carotenoids and serum (Mohammadi et al., ) which contain large quantities of protein, sugar, organic and inorganic salt.
In terms of inorganics, sulfuric acid utilized in coagulation process of skim latex produce high concentration of sulphate in and later discharged as effluent. The sulphate then will produce hydrogen sulphate (H S), during biological anaerobic treatment process. Zinc oxide are used in the vulcanization latex. As a result, it will produce high concentration of zinc in the effluent. Long term exposure of zinc will cause nausea and anaemia. Genetic of aquatic life also will be affected and changed due to the unknown chemicals. Therefore, untreated latex wastewater is not allowed discharge to rivers and streams. Therefore, implementation of proper wastewater management whether biologically or chemically treated is required in order to minimize the pollution and future environmental arising issues (Fakhri et al., , Hena et al., , Liu et al., , Song and Liu, , Sorinolu et al., , Yoo et al., , Zhang et al., ). Wastewater treatment using biological processes is widely used in rubber industries in Malaysia. Pond technology is the most common method being used. Although pond technology can remove % of biological oxygen demand (BOD) from LPW ineffective design and optimum operation, pond technology needed a large area, requires high operating cost and long hydraulic retention time ( -days) (Ibrahim et al., ). Electrochemical method also had been introduced in Malaysia to treat the rubber wastewater. But this method is not successful due to the concomitant energy utilization.
Previously, Atagana et al. ( ) found that effluents from latex processing support microbial growth. The concentrated latex wastewater was identified. The efficiency for organic removal by isolated bacterial was evaluated. Another study showed that, after hours of cultivation, % of chemical oxygen demand (COD) was removed by Rubrivivax gelatinosus and Thiobacillus sp. (Mohammadi et al., ). The purple non-sulphur photosynthetic bacteria (PNSB) isolated from rubber sheet wastewater in Thailand can reduce the chemical oxygen demand and biological oxygen demand of wastewater to % under suitable conditions. Therefore, this study aims to investigate the potential occurrence of viable growth in treated LPW via morphological properties classification base on basic microbiological approach. The effectiveness of a biological wastewater treatment is highly dependent on the viable microorganism community as wastewater nutrient removal involves dynamic and complex biochemical metabolisms either under aerobic or anaerobic conditions.

Materials and methods . Description of real-rubber wastewater treatment plant
Rubber wastewater processing involves various stages which comprises of physical, chemical and biological treatment. Wastewater samples were collected at the equalization tank, chemical treatment process and lastly in final discharge. The wastewater was characterized for physicochemical properties. .

Sample preparation
Samples of wastewater were collected specifically from activated sludge from latex processing effluent. Sample were collected using sterile screw-capped bottle. Sample was stored at a temperature of o C prior to further analysis. .

Inoculation and purification
The entire samples were serially diluted to -prior inoculation. Isolation and purification of bacterial cultures were performed following microbiological standard procedure by using nutrient agar and . % sodium chloride. Pure cultures were then stored in nutrient agar plate and store at refrigerant at o C until needed.

. Motility
A drop of ultrapure water was placed on the centre of the slide. A single isolated colony was taken up from nutrient agar surface then transferred into the water drop and spread by sterilized wire loop. A slide was covered on the drop of water and the motility of bacteria was identified under microscope. .

Fixed smear preparation
A drop of ultrapure water was placed on the centre of the slide. A single isolated colony was taken up from nutrient agar surface then transferred into the water drop and spread the emulsion evenly on the surface of the glass slide to give a relatively thin and smooth layer by sterilized wire loop. The slide was quickly passed through flame of Bunsen burner three times. .

Gram staining
A slide with fixed smear flooded with several drops of crystal violet for seconds, the slide then washed with water to remove excess crystal violet. Gram's iodine was added to cover the smear for seconds. Poured off the iodine and washed through with acetone. De-colorization has occurred when the solvent flows colourlessly from the slide. Acetone then was wash gently with running water tap water. Safranin then covered the smear for seconds.
The safranin then was gently washed for a few seconds; the smear then was blotted and dried at room temperature. Fixed slides then were examined under microscope. .

Bacterial colony classification
Preliminary study of isolates was done by using standard biochemical tests including Gram staining to observe the bacterial morphology properties and motility test for motility behaviour. All the results were then carried out with references to Bergey's Manual of Systematic Bacteriology for classification. Morphology characteristics were based on the characteristic features of bacterial growth in culture. Whole colony, pigmentation, margin and elevation of colony of the bacteria were examined after hours incubated on nutrient agar.

. Analytical Analysis
The wastewater collected were characterized in terms of temperature, dissolved oxygen (DO), Total suspended solids (TSS), Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), heavy metal, ammonia nitrogen, pH, and turbidity according to the standard methods (APHA, ). The temperature is measured by using APHA method and turbidity is measured by using the N turbid meter. While total suspended solid is measured by using HACH method . For the dissolved oxygen, the reading is measured by using YSI model (S/N: F ). While the concentrations of heavy metal and ammonia nitrogen was measured using HACH method.

Result and discussion . Isolation and identification of bacteria
Biological treatment commonly may not be suitable for rubber wastewater treatment. Often, coagulation and flocculation are applied for rubber wastewater treatment. However, this study shows that certain bacteria are able to grow aerobically in treated rubber wastewater indicates their tolerance to rubber wastewater. Seventeen bacterial strains were isolated from collected effluent sample. Out of the isolates purified, were Gram positive and another were Gram negative. Results of microscopic observation and Gram staining were summarized in Table . Collectively, a mixture of certain genus in consortium are effective in nutrient removal significantly in ammonium and phosphate (Pillai and Girish, ). Purple nonsulfur bacteria have shown significant roles in H S elimination in rubber wastewater (Kornochalert et al., ). It was found that certain major genera are significant to rubber processing wastewater. Sulfate-reducing bacteria possessed high growth rate in rubber processing wastewater (Tanikawa et al., ). Promnuan et al. ( ) investigated the potential of Desulfovibrio sp., Desulfitibacter sp., Dethiosulfatibacter sp., and Clostridium sp. in biohythane production and sulfate removal under anaerobic condition. Clostridium sp., Citrobacter sp., Klebsiella sp., and Enterobacter sp.were found possessed optimum growth and capable of producing biohydrogen in rubber latex processing wastewater (Nguyen et al., ). Some genera have shown outstanding removal rate of pollutants. Tanikawa et al. ( ) have discovered key microbes for nitrogen removal in rubber wastewater degradation with highest occurrence of rubberdegrading bacterium Gordonia sp. with . % dominance rate. These bacteria were found to achieve high removal efficiency of residual rubber by both coagulation and biological degradation (Tanikawa et al., ). Pseudomonas sp. is known as indigenous bacteria in rubber wastewater. Recently, Pseudomonas sp. and Stenotrophomonas sp. demonstrated ability to degrade hazardous chemical compound such as -mercaptobenzothiazole (MBT) (Krainara et al., ).

Figure The real-rubber wastewater treatment processes
Rubber wastewater is readily complex with the presence of various chemical compounds used in the processing line. The ability of microorganisms to grow and treat the wastewater is utmost important to ensure minimal environmental impact of the treated wastewater discharged. Recent evidence suggests that there are significant roles of indigenous microorganism in rubber wastewater treatment. Factors found to be influencing their growth, abundance and biodegradability of nutrients and specific compounds in rubber wastewater also have been explored in several studies. This indicates a need to understand the various perceptions of microbial contributions particularly in rubber wastewater. The studies presented thus far provide evidence that there is vast potential in exploration of the biodiversity of microorganisms in rubber wastewater further in depth and to understand their biochemical mechanisms in biodegradation.

Real-wastewater characteristics
The physicochemical characteristics of collected rubber wastewater were determined. The results are shown in Table . The results showed the performance of the rubber wastewater treatment processes according to the real-treatment plants setup. The variation of the wastewater characteristics exhibits the processes occurred in plants subsequent to the manufacturing production segments which involves orderly processes as shown in Figure . At site, the pH was optimized throughout the processes to maintain the treatment requirement. Temperature were not controlled by the operator as the plant were let operated at ambient temperature. The DO started steadily at . , declined to . and raised to . mg/L respectively. Similarly, the BOD and COD were also reduced significantly to from and mg/L to . and mg/L. The biomass showed reduction and it is indicated by the turbidity where it was reduced significantly from to . NTU. Heavy metals i.e zinc, manganese, iron and nickel were analysed accordingly.

Conclusions
Seventeen isolates were successfully isolated from rubberbased wastewater and their morphological properties were determined. Nine Gram-positive cultures and Gram-negative cultures were isolated. The number of isolated cultures accounted % of Gram-positive and % of Gram-negative cultures. Morphological observation allows rapid determination of bacteria in rubber wastewater to indicate viable cell growth. Based on these preliminary results, it can be concluded that some microorganisms always existed in the activated sludge collected from rubber wastewater. This concludes that rubber wastewater consists of high microbial diversity. However, the microbial abundance and bacterial identification shall be further validated using molecular technique.

Declaration of competing interest
The authors declare no known competing interests that could have influenced the work reported in this paper.