Caffeine (1, 3, 7-trimethylxanthine), a well-known alkaloid, is found in natural products such as coffee beans, tea leaves, etc and used by human being in the form of tea, coffee, pharmaceutical and cosmetic products along with in many other forms. In limited amount, it is beneficial for humans’ health, as it can rejuvenate the body and restore alertness by stimulating central nervous system [1]. Worldwide, the average consumption of caffeine is ranging from 80-400 mg per day, and the mean daily intake of 227 mg/day by approximately 90% of the adults worldwide [2]. Such consumed caffeine becomes a part of environment through the excretory residues and the domestic effluents, by the leaching of caffeine from the expired pharmaceutical and cosmetic products deposited in dumping sites, through the effluents of manufacturing industries, hospital wastes etc [3]. Through all these processes, caffeine becomes a part of surface and groundwater bodies and can also contaminate the soil. Caffeine is considered as chemical marker for pollution and it is having high water solubility (13 g/L), low volatility and have half-life of approximately 10 years [4]. Caffeine present in soil can have negative effect on the germination of seeds and seedling growth. The microbes such as Pseudomonas sp., Klebsiella, Rhodococcus sp. Serratia etc are reported to have the caffeine degradation capacity [5]. So bioaugmentation using such microbial strains can accelerate the degradation of caffeine in soil.

As Pseudomonas sp. (GBPI_Hb5) had shown high caffeine degradation capacity in aqueous medium, the same bacterial strain was tested for analyzing its caffeine degradation capacity in soil media. For this, the soil was collected locally. The soil sample was divided in to two parts, one part was autoclaved in autoclavable bags at 121°C for 20 min while other remained unsterilized. Both types of samples were impregnated with caffeine by mixing with 1000 mg/L caffeine aqueous solution in the ratio 1:1 (w/v), in an autoclaved container. The Pseudomonas sp. (GBPI_Hb5) bacterial strain solution was cultured in tryptone yeast medium (TYE) up to 24 h when the optical density of the solution had reached 0.75 units. The sterilized and unsterilized soil samples were mixed with this bacterial solution in the ratio 2:1 (w/v) under sterile conditions. These bacterial and caffeine impregnated soil samples were used for the caffeine degradation experiments. 60% moisture was maintained in the soil during the experiments

100 g soil samples were taken in pot tray in triplicate for both the sterile and unsterile soil samples and incubated for 65 days under ambient conditions. Separate sets for different durations were arranged including 0, 1, 5, 10, 15, 25, 35, 45, 55, and 65 days for caffeine biodegradation study. Approximately 10 gm soil samples were taken from respective pots and mixed with 20 mL methanol (HPLC grade). The mixture was agitated for 30 min in incubating shaker, and then filtered using Whatman filter paper 1 for the separation of soil particles. The supernatant was again filtered using membrane filter (0.2 μm nylon 66) for the removal of bacterial strains, if any. The filtrate was analysed using UV-Vis-spectrophotometer (Shimadzu UV 2600) at 273 nm and the quantification of caffeine was done using calibration curve.

The presence of Pseudomonas sp. (GBPI_Hb5) in both the sterile and unsterile soil had significantly increased the caffeine removal from the artificially contaminated soil samples (Figure 1). In sterile soil, the maximum caffeine removal was 75% while in unsterile soil the maximum caffeine removal was 91%, which might be due to combined effect of Pseudomonas sp. (GBPI_Hb5) and the bacterial strains already present in the unsterile soil as without the targeted bacteria, the caffeine degradation was only 7%.

While studying the caffeine degradation, kinetics of the process was also tried to be studied using duration and caffeine concentration at time t data. The calculated kinetic parameters for caffeine degradation in the experiments were shown in Table 1. The data from the kinetic model suggested that the degradation of caffeine under the ambient condition followed first-order kinetics, where the k values range from 0.0011 to 0.0215 day^-1 and the degradation ranges from 6.8 to 91% in 65 days for 1000 mgL^-1 caffeine concentration. The regression coefficient of degradation of caffeine ranges from (0.8005 to 0.931) showing that data of degradation were correlated well with the kinetics model. The control of the sterilized and non-sterilized soils for t_1/2 was observed at 630 and 990 days, whereas, in the treated soil with bacterial strain GBPI_Hb5, the values of t_1/2 were decreased significantly to 34.5 and 32.2 days, respectively. The above-mentioned results confirmed that the bacterial strain GBPI_Hb5 is an ideal bacterial strain for the biodegradation of caffeine-contaminated sites. Cycon et al. [6] reported the deltamethrin degradation in bio augmented soil using Serratia marcescens bacterial strain. The authors reported that the deltamethrin contamination bioaugmentation in the non-sterile soils using microorganisms increased the degradation potential pyrethroid and the value of t_1/2 was reduced significantly in different types of soils (i.e., sandy, sandy, silty loam, silty) respectively. Bhatt et al. [7] reported that the bacterial strain CW3 was also degraded allethrin in natural soil environments with moderately less t_1/2 values, which indicates the potential of bacterial strains in the pyrethroid bioremediation in the contaminated environment. The microorganisms reported for the biodegradation of target compound are responding properly in laboratory conditions but their behaviour might be different in natural conditions due to biotic and abiotic stresses in the natural conditions [8], which need to be studied.

This study shows the potential of GBPI_HB5 in the natural soil environment. Bioaugmentation studies related to soil with the bacterial strain GBPI_HB5 confirm the t_1/2 value significantly decreases in presence of the bacterial strain used for the study. The bacterial strain GBPI_H5 efficiently degrades caffeine is useful to develop the environment-friendly process for decaffeination. The present information for biodegradation of caffeine in the natural environment should be very useful to the onsite biodegradation in the caffeine contaminated area.

The authors are grateful to Director GBP-NIHE for extending the facilities and the Department of Science and Technology-Water Technology Initiative (DST-WTI) for financial support.