All soil, sludges and leachate samples showed growth in LB media, which confirmed the existence of bacterial colonies inside all the samples, however, only the soil samples exhibited growth in the acidic 9k-Fe media. This observation shows the tolerance of the bacterial colonies in the samples to the acidic conditions, which can be further inoculated for bioleaching. Soil 1 performed the best during the bioleaching screening using the exposed copper wPCB. The usage of this kind of wPCB allowed the one-step bioleaching mechanism to be applied, as the toxicity of wPCB due to its components can be neglected because it was a blank PCB. The significant amount of copper extracted from the wPCB using Soil 1 as the bacterial consortia might occur due to the presence of more colonies capable of copper bioleaching than the other three sources, thus, Soil 1 was used as the bacterial source to further isolate individual colonies for wPCB bioleaching.
Soil 1 was used as a source for bacterial isolation obtaining four individual colonies, SE, SE2, SC and S1A. The four colonies were submitted for partial 16S rRNA gene sequencing for identification. Strain SE was identified as Bacillus sp. with 99% similarity to Bacillus bingmayongensis. B. bingmayongensis is known to be able to grow in a wide range of conditions, including low pH conditions, as it was reported to be able to adapt to the environment in which it was found and grow at a pH of approximately 2–12 (Liu et al. 2014). The strain B. bingmayongensis is closely related to B. cereus, which has been reported to be able to perform bioleaching of mica from kaolin (Zaremba and Smoleński 2000) and participate in the biosorption of Zn2+ (Joo et al. 2010). Despite the low copper extraction performance of this strain (1.04 ppm), no previous study was reported on the ability of this strain for copper bioleaching.
Strain SE2 was identified to be similar to Lysinibacillus boronitolerans, with a similarity of 98.81%. This species is known to be widely available in soil with no mention of its ability to grow under low pH conditions (Nam et al. 2012). The present study, however, was able to isolate the strain under low pH (pH = 2.50) conditions. The strain was reported to show metal-binding properties and remediation toward contaminated matrices (Bustos et al. 2018). Similar to strain SE, strain SE2 also shows low copper bioleaching ability, with copper recovery of 9.16 ppm. This is also the first report on the use of this strain as a bacterial strain for copper bioleaching.
Strain S1A was identified to be similar to Oryzobacter terrae, with a 96.55% similarity. There is no previous report on the ability of this strain to perform bioleaching, and this strain performed the worst during the bioleaching procedure with only 0.62 ppm of copper extracted.
Strain SC was identified to be in the same genus as strain SE, Bacillus sp., but yielded a better bioleaching result, with a 182.95% difference. Besides, this strain was identified to have 98.23% similarity to B. terrae. Similar to strain SE, this strain has never been reported to be involved or to have any abilities to perform bioleaching of copper or any other metal. The present research, however, was able to analyze and identify that the bioleaching of copper can be performed using this strain. Compared to a previous study, we were able to grow this strain at a low pH rather than at pH 5 to 8 and optimum at pH 7 (Díez-Méndez et al. 2017).
All strains displayed clear physical changes in the leaching media during bioleaching. Brown precipitates were formed due to the oxidation of Fe (II) to Fe (III), which was present inside the media. Similar to the proposed ionic exchange in the bioleaching mechanism, these changes were reported in a previous study (Hansford and Vargas 1999; Zhang et al. 2013). The color of the media also changed from a pale shade of green (due to Fe (II) initially in the media) to a shade of blue, indicating the presence of Cu (II) inside the media.
The solubilization of copper by strain SC was evaluated by using copper strips to determine the interaction of the strain during copper solubilization. From the 1g copper strip used, 0.80 mg/g (± 0.02) was solubilized, as analyzed using AAS in comparison to the set control with only 0.03 mg/g (± 0.08) solubilized in the absence of strain SC. By comparison, a previous study reported, the total amount of copper extracted through bioleaching to be approximately 80–90% within a similar timeframe used in the current study (Rodrigues et al. 2015). This result indicates that strain SC has the ability to solubilize copper, thus confirming the activity of the strain shown during the bioleaching process with wPCB.
As this study focused more on the novelty of bioleaching as a standalone process to limit other procedures that involve either heavy mechanical or pyrometallurgical processes, the present research managed to minimize the use of mechanical methods, such as grinding down the wPCB to a size smaller than 0.55 mm (Zhang et al. 2013; Shirodkar and Terkar 2017; Xia et al. 2017), which produces microdust as the byproduct of the mechanical grinding process and generates secondary pollutants from the process itself. Minimal mechanical treatment helps to retain the wPCB in the original shape after bioleaching and allows the copper to be extracted via solubilization of the metal itself, producing less toxic leachate than the previous study. In fact, copper bioleaching using the standalone process have successfully been performed by all the isolated bacterial strains, though the performance of each strains were varied.