A high-efficiency Klebsiella variicola H12-CMC-FeS@biochar for chromium removal from aqueous solution

In polluted groundwater, surface water, and industrial sites, chromium is found as one of the most common heavy metals, and one of the 20 main pollutants in China, which poses a great threat to the ecological environment and human health. Combining biological and chemical materials to treat groundwater contaminated by heavy metals is a promising restoration technology. In this research, Klebsiella variicola H12 (abbreviated as K. variicola) was found to have Cr(VI) reduction ability. A high-efficiency Klebsiella variicola H12-carboxymethyl cellulose (abbreviated as CMC)-FeS@biochar system was established for Cr(VI) removal from aqueous solution. The Scanning Electron Microscope-Energy Dispersive Spectrometer (SEM–EDS), X-ray photoelectron spectroscopy (XPS) results indicated that CMC-FeS was successfully loaded onto the surface of biochar, and K. variicola H12 grew well in the presence of CMC-FeS@biochar with microbial biomass up to 4.8 × 108 cells mL−1. Cr(VI) removal rate of CMC-FeS@biochar system, K. variicola H12 system and K. variicola H12 + CMC-FeS@biochar system were 61.8%, 82.2% and 96.6% respectively. This study demonstrated K. variicola H12-CMC-FeS@biochar system have potential value for efficient removal of Cr(VI) from Cr(VI)-polluted groundwater.

K. variicola H12. Klebsilla variicola H12 was obtained from our laboratory, which was screened from sludge in a previous work 1 , Based on the blasted results of the 16S rDNA gene sequence, this novel strain was named as Klebsiella variicola H12. It was preliminarily identified by using physiological and biochemical analysis including colony morphology, Gram staining, methyl red (MR), oxidase, Voges Proskauer test (VP), nitrate reduction, malonate utilization. The experiment was based on LB medium(10 g L −1 Tryptone, 5 g L −1 yeast extract, 10 g L −1 NaCl), with different carbon sources (C 6 H 12 O 6 , C 12 H 22 O 11 , (C 6 H 10 O 5 ) n , C 2 H 3 O 2 Na·3H 2 O), different nitrogen sources (Beef Extract, Peptone, (NH 4 ) 2 SO 4 , CH 4 N 2 O) and different organic salts(NaH 2 PO 4 , KH 2 PO 4 , NH 4 H 2 PO 4 , Na 3 PO 4 , Nacl) as medium components. The energy substrate experiment was performed with 150 rpm at 30 °C for 24 h. In addition, the experiments set gradient pH (2.0, 4.0, 6.0, 8.0, 10.0) and temperature (20 °C, 30 °C, 40 °C, 50 °C, 60 °C) in LB medium at 150 rpm. The incubation time was the same as above. The above experiments were set up in two parallel groups.
Removal of Cr(VI) by K. variicola H12. K. variicola H12 was cultured with 150 rpm at 30 °C in LB medium for 20 h to obtain late logarithmic phase cells. 10 g L −1 Tryptone, 5 g L −1 yeast extract, 10 g L −1 NaCl were prepared into a liquid medium, and the pH value was adjusted to 6.0. In the experimental group, the mother solution of K 2 Cr 2 O 7 with a concentration of 2000 mg L −1 was added to the LB medium, so that the final concentration of Cr (VI) in the medium reached 20 mg L −1 , 60 mg L −1 , 100 mg L −1 , 200 mg L −1 . Control group was cultured in the medium without the mother solution of K 2 Cr 2 O 7. 1 mL samples were taken out every two hours, and the cell density of K. variicola H12 was measured with an Epoch Microplate spectrophotometer (BioTek Instruments, Inc), at OD 600 (optical density at 600 nm) 36 . Then, the concentration of Cr(VI) in sample was determined by 1, 5-diphenyl carbazide method 37 . Total chromium was first oxidized by potassium permanganate and then measured. Specifically, 0.2 g of 1,5-diphenylcarbazide was dissolved in 50 mL acetone, then 50 mL of deionized water, 12.5 mL of 85% phosphoric acid and 12.5 mL of 95% concentrated sulfuric acid were mixed with www.nature.com/scientificreports/ the solution to obtain a chromogenic reagent. Determination of total chromium is performed by adding 40 g L −1 KMnO 4, 200 g L −1 (NH 2 ) 2 CO and 20 g L −1 NaNO 2 before that step. 3.0 mL of the chromogenic reagent was added to the as-mentioned supernatant and the mixed solution was tested at 540 nm on a spectrophotometer.

Preparation of CMC-FeS@biochar. CMC-FeS@biochar was prepared following the methods used by
Lyu et al. 25   Analytical methods. The late logarithmic phase cells were centrifuged at 8000 rpm for 5 min, and then washed three times with phosphate buffer. The morphologies of bacterial cells and CMC-FeS@biochar were observed by XL 30ESEM scanning electron microscopy (SEM) (Hitachi S4800) 39 . The energy dispersive spectroscopy (EDS) at a voltage of 150 keV was also operated to identify the chemical elements on cell and CMC-FeS@biochar surface. In addition, before adsorption and after adsorption of Cr(VI), the functional groups was analyzed by the Fourier transform infrared spectrophotometry (FTIR spectrum, Nexus670, Thermo Nicolet Co.) with the wavelength range of 400-4000 cm −1 . X-ray photoelectron spectroscopy (XPS) of CMC-FeS@biochar was done before adsorption of Cr(VI). The XPS spectra operated on a Thermo Fisher-VGScientific (ESCA-LAB 250Xi) photoelectron spectrometer. The surface area and pore structures of CMC-FeS@biochar were determined by the BET adsorption-desorption method (version 11.02; Quantachrome Instruments, USA).

Results and discussion
Klebsiella variicola H12. Physicochemical property of K. variicola H12. The Physicochemical property of K. variicola H12 and the utilization of energy substrates are shown in Fig. 1. When culture medium was pH 6.0 and the temperature was 30 °C, the growth of the strain was the best which OD 600 reached around 1.6 ( Fig. 1b 4 , Nacl to meet the needs of spontaneous growth. However, in the culture medium that tryptone as nitrogen sources, yeast extract as the carbon source, and NaCl as the inorganic salt, the strains grew best and could be used as the culture medium for subsequent experiments. SEM-EDS analysis of K. variicola H12. The analyses of SEM-EDS were adopted to investigate the cell morphological changes of K. variicola H12 before and after treated with 20 mg L −1 Cr(VI) for 24 h (Fig. 1c-f). It can be seen that in the Cr(VI) -free culture, the cells were short rod shaped [size (0.5 ~ 1) μm × (1 ~ 2) μm] and the surface was depressed (Fig. 1c). According to EDS analysis (Fig. 1d), C, N, O were the main elements of K. variicola H12. After adding Cr(VI) to the culture medium, the morphology of K. variicola H12 cells changed (Fig. 1e). Large number of cells were covered with a certain substance. EDS analysis (Fig. 1f) further proved that the main elements on the surface of these substances contained C, N, O and Cr is also detected. It was speculated to be some organics such as Extracellular polymers secreted of strain after being stimulated by chromium [40][41][42][43] . This was one of the stress response of strains to the surrounding environment.
Cr(VI) reduction by K. variicola H12. The effect of different concentrations of chromium on the growth of K. variicola H12 is shown in Fig. 1g (Data provided in Table S3). From the change rule of OD 600 , it can be seen that the value of OD 600 gradually decreased with the increase of Cr(VI) concentration in the same culture time. It indicated that the biotoxicity of metals increased with the increase of metal concentrations. In the absence of Cr(VI) solution, the bacteria have the highest activity in the culture medium, and the OD 600 reaches 1.4 after 20 h of culture. Before this period, cell synthesis and metabolism were very rapid due to sufficient nutrients in the LB medium and no toxic effects of heavy metals. When the Cr(VI) concentration increased from 20 to 100 mg L −1 (20 mg L −1 , 60 mg L −1 , and 100 mg L −1 ), the maximum OD value decreased from 1.2 to 0.2. When Cr(VI) concentration reaches 200 mg L −1 , K. variicola H12 did not grow. It illustrated that the K. variicola H12 could survive in Cr(VI) concentration of < 200 mg L −1 , which proved that the strain has the ability to tolerate Cr(VI). The toxic effect of heavy metals in the solution increased with the gradual increase of chromium concentration and the growth and reproduction ability of the strain decreased. Under high Cr(VI) concentration, the growth and reproduction of K. variicola H12 are inhibited. In addition, In order to verify that K. variicola H12 has ability to remove Cr(VI) from liquid culture medium, the concentration of Cr(VI) in 20 mg L −1 culture medium was analyzed every 2 h. As shown in Fig. 1h (Data provided in Table S4), the reduction rates of Cr(VI) were 58.96%, 86.79% and 93.87% at 4 h, 8 h and 12 h after bacterial cells treated. At any time point, the total chromium content that can be detected in the solution was higher than the Cr(VI) content. It indicated that Cr(III) was present in the solution, the concentration Cr(III) provided in Table S4. Cr(III) may be derived from the reduction of Cr(VI) by reductase which produced by bacteria in the growth and metabolism of Cr(VI) environment.  (Fig. 2b) respectively, and the satellite between the two dominant peaks is contributed by Fe 2+ through the process of oscillation 46,47 . The binding energies of 169.1 eV and 160.9 eV represent S 6+ 2p 3/2 and S 2-2p 3/2 45,48 (Fig. 2c). It also indicated that FeS was successfully attached onto the surface of biochar. In addition, as shown from Table 2 (BET surface area calculated in the relative pressure region P/P0 = 0.300, Micropore volume determined at P/P0 = 0.991, Average pore diameter obtained from BJH equation using N 2 isoterms). After adsorbing Cr(VI), micropore volume and average pore diameter decreased, which indicated the existence of pore filling effect during the process.

Characterization of K. variicola
SEM-EDS analysis of K. variicola H12-CMC-FeS @ biochar system. SEM-EDS analysis of the bare biochar, CMC-FeS@biochar, K. variicola H12-CMC-FeS@biochar composite are shown in Fig. 2d-k. Morphological observations shown that the biochar (Fig. 2d.) was porous and rough, that resulted in more uniform attachment of FeS and creation of larger specific surface area and pore volume yielding more sorption sites. Compared with biochar, clearly defined aggregated particle was observed on the surface of biochar (Fig. 2f). It indicated that irregular granular like CMC-FeS@biochar particles in diameter of < 100 nm were attached to the biochar surface. The analysis of EDS was adopted to investigate the elemental in CMC-FeS@biochar composite. As seen from Fig. 2e, C, O, Si, Ca were detected by EDS which should be ascribed to the components of the biochar. After supporting, the element Fe and S were detected (Fig. 2g). The successful loading of FeS on the surface of biochar was initially proved. In K. variicola H12-CMC-FeS @ biochar system, SEM image of K. variicola H12-CMC-FeS@ biochar shown in Fig. 3e,f. The K. variicola H12 was unevenly attached to the surface of the CMC-FeS@biochar (Fig. 2h). As seen from Fig. 2i, when a large amount of K. variicola H12 was adsorbed on the surface of the material, EDS analysis of the surface of the material detected the presence of N element. It indicated that CMC-FeS@ biochar has formed a complex with K. variicola H12. As shown in Fig. 2k, chromium was found on the surface of CMC-FeS@biochar. It indicated that there is an adsorption effect between CMC-FeS@biochar and chromium.

Removal of Cr(VI) by K. variicola H12-CMC-FeS@biochar system. Effect of CMC-FeS @ biochar on
the growth and tolerance of K. variicola H12. The effect of CMC-FeS @ biochar on the growth and tolerance of K. variicola H12 are shown in Fig. 3. The growth of K. variicola H12 on LB medium was assessed in the presence of CMC-FeS@biochar (Fig. 3a) (Data provided in Table S5). The stabilization of CMC (CMC-FeS) and supporting of biochar (CMC-FeS@biochar) reduced the toxicity of FeS, as evidenced by increasing the growth of K. variicola H12 from OD 1. 39 25 . Bare FeS has significant inhibitory effect on bacterial 25 and the study of Lee et al. also reported that bacterial growth was restricted by iron nanoparticles through physical coating, oxidative stress, and membrane disruption 49 . The effect of CMC-FeS@biochar on the tolerance of K. variicola H12 is shown in Fig. 3b (Data provided in Table S6). At a lower Cr(VI) concentration (20 mg L −1 ), the effect of CMC-FeS@biochar on the growth of bacteria was significantly higher than that of high concentrations obviously. When CMC-FeS@biochar was present, the highest cell concentration of bacteria can reach to 4.8 × 10 8 cells mL −1 . It indicated that CMC-FeS@biochar promoted the growth of K. variicola H12 and lay the foundation for the efficient removal of Cr(VI) in groundwater.

Performance of K. variicola H12-CMC-FeS@biochar system in removing Cr(VI). Cr(VI) can be removed by K.
variicola H12-CMC-FeS@biochar system (Fig. 3c) 15 . While in reduction, the removal mechanism of Cr(VI) mainly relied on the reducibility of Fe 2+ and S [2][3][4][5][6][7][8][9][10][11][12][13][14][15] . The dominant removal mechanisms of K. variicola H12 on Cr(VI) was confirmed in 3.1 (Fig. 1h). In addition, a small amount of surface adsorption has also been confirmed in 3.1 (Fig. 1e,f). In the bacteria + CMC-FeS@biochar system, the removal efficiency of Cr(VI) was improved, compared to the CMC-FeS@biochar and single bacteria treatment system.  www.nature.com/scientificreports/ These showed that in the mixed system, CMC-FeS@biochar and K. variicola H12 have a synergistic relationship with each other when processing Cr(VI). But it is not a superposition of efficiency. Compared with chemical group [Cr(VI) removal efficiency were less than 62.3%] and the bacteria mixed with chemical group (the Cr(VI) removal efficiency of K. variicola H12 + biochar, K. variicola H12 + FeS reached 80.8%, 79.5% respectively), the bacteria + CMC-FeS@biochar system removal rates of Cr(VI) increased by 1.6 times and 1.2 times respectively. In the 6th hour, the removal rate of Cr(VI) reached the highest value first. When the same amount of heavy metal Cr(VI) was processed, the other experimental groups had a longer processing time and a relatively low removal rate. At the same time, Compared with the research by Liu et al. 50 (Who used Cr (VI) resistant bacterial strains to treat chromium-containing sludge, the Cr (VI) level was decreased by 90% within 65 h, the processing time is shortened by about 1/6, and the efficiency is 1.05 times of them in this study). In situ remediation, nZVI and other chemical methods were used by some researchers and the efficiency can reach more than 95% 51 . However, the extensive use of these chemicals caused secondary pollution to the surrounding environment. The superiority of K. variicola H12-CMC-FeS@biochar system to treat Cr (VI)-contaminated groundwater was confirmed. For further application of K. variicola H12-CMC-FeS@biochar in wastewater treatment, a regeneration study was performed in this work. There was a gradual decrease in Cr(VI) desorption rate with an increasing number of cycles as shown in Fig. S1. The desorption rate of cycles 1, 2, 3 and 4 were found to be 61.5%, 40.6% 26.7% and 11.2% respectively. It was observed that cycle 1 showed good retrieval. Cr(VI) removal decreased from 96.6% in LB medium to 50.4% in real water sample (Fig. S2). It is speculated that some inorganic and organic substances in the real water sample have an inhibitory effect on the removal of chromium 52 . , it can be further speculated that in the process of processing chromium, there may be a cooperative processing relationship between K. variicola H12 and CMC-FeS@biochar. Comparing the spectra of biochar and K. variicola H12-CMC-FeS@biochar, K. variicola H12-CMC-FeS@biochar provided more functional groups for binding Cr(VI) and has a better ability to loading Cr(VI).

Effects of inoculation amount, oxygen condition and solution pH on Cr(VI) removal.
In this experiment, the K. variicola H12-CMC-FeS@biochar system was further studied. The effects of inoculation amount, oxygen, and pH on the K. variicola H12-CMC-FeS@biochar system to remove Cr (VI) are shown in Fig. 4. Figure 4a (Data provided in Table S8) showed that at the 6th hour of treatment, the inoculation amount of 10% (which had a removal rate of 95%) was higher than the inoculation amount of 2% (which had a removal rate of 81%). It indicated that the inoculation amount of K. variicola H12 had a certain effect on the efficiency of Cr(VI) removal by K. variicola H12-CMC-FeS@biochar system. The larger the inoculation amount, the higher the processing efficiency. These was consistent with the results of Liu et al. (2019) study 50 . Cr(VI) was removed more efficiently under anaerobic conditions, relative to aerobic conditions (Fig. 4b) (Data provided in Table S9). Under the condition of pH 6.0, the removal of Cr(VI) is slightly more efficient than pH 4.0, pH 8.0 (Fig. 4c) (Data  Table S10).These experiments showed that different environmental conditions (inoculation amount, oxygen, pH) can affect the efficiency of the K. variicola H12-CMC-FeS@biochar system in the treatment of Cr(VI), which needs to be considered in practical application. In addition, SEM analysis shown that there are some differences in the stability of K. variicola H12-CMC-FeS@biochar under different acidic and alkaline conditions ( Fig. 4d-f). In the pH 4.0 and 8.0, the amount of K. variicola H12 was relatively small compared to that in the pH 6.0. Therefore, the K. variicola H12-CMC-FeS@biochar system performs well under the conditions of anaerobic, 10% inoculation amount and pH 6.0.

Discussion
A groundwater remediation method for Cr (VI) removal using K. variicola H12-CMC-FeS@biochar was developed. CMC-FeS@biochar was successfully synthesized and utilized in the effective Cr (VI) removal from Cr(VI)contaminated aqueous solution. K. variicola H12 can tolerate 100 mg L −1 of Cr(VI). The CMC-FeS@biochar increased the growth of K. variicola H12 at late logarithmic phase, though it decelerated the growth of K. variicola H12 at lag phase. With the addition of K. variicola H12, Cr(VI) removal rate of K. variicola H12-CMC-FeS@ biochar system reached 96.6% for 10 h and the variety of surface functional groups increases, which provides more binding sites. K. variicola H12-CMC-FeS@biochar had a synergistic relationship with each other, but it was not a superposition of efficiency. The optimal conditions for the growth of K. variicola H12 are pH 6.0 and 30 °C. Tryptone, yeast extract and NaCl used as nitrogen sources, carbon source and inorganic salt were used by K. variicola H12. When Cr (VI) concentration arrived at 200 mg L −1 , the growth of K. variicola H12 strain was completely inhibited. XPS and FTIR analysis showed that CMC-FeS was successfully loaded on biochar. Then K. variicola H12-CMC-FeS@ biochar was successfully constructed. In this system, the maximum OD value of K. variicola H12 was promoted by CMC-FeS@biochar from 1.39 to 1.57 and the removal rate of Cr(VI) was reached 96.6% at 10 h. This was shorter than the previous study (36 h is required to process chromium for microbial-biochar) 59 . FTIR analysis shown that K. variicola H12-CMC-FeS@biochar has more functional groups on the surface to provide bonding Site. Under different acidic and alkaline environments, EDS showed that the K. variicola H12-CMC-FeS@biochar system remained stable except for different strains (K. variicola H12 grew best under neutral conditions).
In this study, K. variicola was found to have the ability to remove chromium, which provided a theoretical basis for K. variicola H12 to be used in groundwater pollution of heavy metals. In addition to the existing traditional chemical treatment methods, combining microorganisms (K. variicola H12) with CMC-FeS@biochar can remove Cr(VI) more efficiently, which provides a new idea for groundwater treatment methods. At the same time, the biochar formed by pyrolysis of waste wheat straw was used as the main load material, which has advantages in environmental protection and economy. It has the potential for practical application.