International Journal of Environmental Science and Technology https://doi.org/10.1007/s13762-022-04000-6 ORIGINAL PAPER Insights of microorganisms role in rice and rapeseed wastes as potential sorbents for metal removal X. Shen1,2 · J. Zhao2,3 · N. Bonet‑Garcia4 · E. Villagrasa4 · A. Solé4 · X. Liao1 · C. Palet3 Received: 5 August 2021 / Revised: 20 January 2022 / Accepted: 25 January 2022 © The Author(s) 2022 Abstract Rice and rapeseed agricultural wastes, as nonliving biomass, are proposed for heavy metal remediation in polluted effluents (chromium, cadmium, copper and lead). The physicochemical characterization of these biomasses shows that the surface of both sorbents is negatively charged (zeta potential), the surface area of sorbents is 4.39 and 40.7 (Brunauer–Emmett– Teller), and the main functional groups are carboxylic and hydroxyl (attenuated total reflection Fourier-transform infrared spectroscopy). The main purpose of this work is to evaluate the insights of microorganisms associated with these nonliving biomasses in the removal of heavy metals from synthetic aqueous solutions, adjusted at pH 4.0 (as the best acidic condition for the sorption process). The isolates (Bacillus genus in rice and Escherichia, Micrococcus and Staphylococcus genus in rapeseed) remove heavy metals from mentioned solutions, mainly in consortia, with contribution percentage over than 80% of total metals. In addition, when they are present in biomass, they provide an additional metal removal effect, especially in rapeseed biomass system and with multiple heavy metals aqueous solutions: i.e. Cr(III) removal, at 4 mmol/L, increases from 70 to 100%. This knowledge makes possible the use of the nonliving biomasses with no need for any special pretreat- ment against the microorganisms, prior to their use as metal sorbents that implies their good feasibility for application from an economical point of view. Keywords Biomass waste valorization · Sorption · Uptake heavy metals · Bacteria Introduction Heavy metal (HM) pollution has mainly become a serious Editorial responsibility: Maryam Shabani. threat to the environment due to anthropogenic activities (Bhattacharya et al. 2015). This increase in attention is due X. Shen and J. Zhao have contributed equally. to the high toxicity, persistence in sediment and accumula- tion in biological tissues of HMs (Martín et al. 2015). HMs * C. Palet cristina.palet@uab.cat can be incorporated into the food chain, which can lead to gastrointestinal, neurological, haematic and renal diseases, 1 Oil Crops Research Institute of Chinese Academy among others (Kim and Kan 2016; Zhao et al. 2020). Conse- of Agricultural Sciences/Key Laboratory of Biology quently, the world is facing a critical water quality problem, and Genetic Improvement of Oil Crops of the Ministry of Agriculture, Wuhan 430062, China which is threatening to animals and human beings (Azizul- 2 lah et al. 2011). Relatively modest concentrations of Cr(III), Key Laboratory of Optoelectronic Devices and Systems, College of Physics and Optoelectronic Engineering, Cd(II) and Pb(II) have toxic effects on the environment and Shenzhen University, Shenzhen 518060, China humans. Cu(II) is also a potential toxicant at high doses 3 GTS‑UAB Research Group, Department of Chemistry, (Tchounwou et al. 2012). Facultat de Ciències, Universitat Autònoma de Barcelona, The main heavy metal pollutants and their main sources 08193 Cerdanyola del Vallès, Catalunya, Spain have included fertilizer leaching, sewage discharge, indus- 4 Department of Genetics and Microbiology, Facultat trial wastewater and urban construction over the last dec- de Ciències, Universitat Autònoma de Barcelona, ades (Nguyen et al. 2013; Addel-Ghani and El-Chaghaby Edifici C‑Campus de La UAB, Bellaterra, 2014; Luo et al. 2017). In particular, the pollution of water 08193 Cerdanyola del Vallès, Catalunya, Spain 13 Vol.:(0123456789) International Journal of Environmental Science and Technology resources by HMs is an alarming issue in China, where large attenuated total reflection Fourier-transform infrared (ATR- amounts of HMs have been found in groundwater and drink- FTIR), zeta potential and Brunauer–Emmett–Teller (BET) ing water (Dai et al. 2018). techniques are employed to identify the properties of the Conventional physicochemical techniques for treating assayed nonliving biomass (type of functional groups and effluents laden with HMs include ion exchange, solvent electronic charge present on the surface of materials along extraction, electrochemical treatment and membrane filtra- with the area, volume and morphology of materials), and (ii) tion technologies (Yin et al. 2019). These methods are usu- the adsorption capabilities of the nonliving assayed biomass ally too expensive and not eco-friendly due to the liberation are compared and studied with/without microorganisms via of toxic chemical sludge that can cause secondary pollution batch adsorption experiments. With this purpose, an eco- (Carolin et al. 2017; Kumar and Khan 2021). Therefore, nomical and natural alternative/substitute for the decontami- using low-cost biosorption approaches for the removal of nation of HM-containing effluents is developed by taking harmful heavy metals from water resources and wastewaters advantage of RO and LO straw residues. Moreover, to the is one of the most important goals in environmental science best of our knowledge, this paper is the first to describe the (Zhao et al. 2019; Zhang et al. 2020; Zhao et al. 2020). One role of microorganisms isolated from LO and RO wastes in of the types of biosorption HM removal technologies, which regard to HM sorption capacity. consists of the use of nonliving biological materials, has recently emerged as a cost-effective, eco-friendly and simple alternative to remove toxic HMs from water or to render Materials and methods these pollutants harmless (Senthil Kumar and Gunasund- ari 2018; Calderón et al. 2020). Therefore, nonliving and Materials and solutions metabolically inactive biomaterials from plants have been increasingly studied (Jain et al. 2015; Zhao et al. 2020). In Rice (Oryza sativa L., RO) and rapeseed (Brassica napus L., particular, rice (Oryza sativa L., RO), which is the staple LO) were provided by the Oil Crops Research Institute of food for more than 50% of the world's population (Zhou the Chinese Academy of Agriculture (Wuhan, China). Both et al. 2014), and rapeseed (Brassica napus L., LO), which is biomasses were first immersed in a clean water solution, an oilseed crop and the second largest source of vegetable phases were separated by decantation, and then, biomasses oil (FAO 2014), are both widely planted in Hunan Province, were ground and dried for further use in corresponding China. RO and LO have been widely studied in environ- metal sorption experiments. All chemicals were of analytical mental fields, in which RO and LO crop varieties have been grade. Stock solutions (approximately 1000 mg/L) were pre- planted in contaminated soils for heavy metal uptake and pared by dissolving the required amounts of Cr(NO3)39H2O, soil remediation (Cárdenas-Aguiar et al. 2020; El-Hassanin Cu(NO3)23H2O, Cd(NO3)24H2O and Pb(NO3)2 (all 99% et al. 2020). After harvesting, nonliving RO and LO residues from Panreac, Barcelona, Spain). (biomass), such as their husks (Zhan et al. 2020) and straw compost (Yu et al. 2020), have been studied as potential Characterization adsorbents for the removal of heavy metals from contami- nated water. Otherwise, it is largely known that living organ- Physicochemical characterization isms (bacteria and others) are generally exposed to mixtures of metals in the environment (Vijver et al. 2011; Mebane RO and LO nonliving biomasses have been properly char- et al. 2020). Hence, bacteria show resistance mechanisms acterized. Therefore, the physicochemical properties of the such as extracellular sequestration, intracellular sequestra- biomass, including the pH, mineral composition, surface tion, active export and enzymatic detoxification, which help area and surface charge, along with the identification of them interact with metals and tolerate their toxicity and del- functional groups, can help to explain the process of metal eterious effects (Yin et al. 2019; Villagrasa et al. 2021). sorption. Attenuated total reflection Fourier-transform infra- Despite the extensive information mentioned above, red (ATR-FTIR, Tensor 27, Bruker, USA) spectroscopy was nothing is known about the role of RO- and LO-associated performed to identify the chemical functional groups pre- microorganisms. Consequently, the aims of this work are (i) sent on the biomass wastes used as sorbents. The FTIR data to quantify the HM sorption potential of nonliving RO and were obtained from 600 to 4000 ­cm−1 with an average of LO biomass in an HM-contaminated aqueous solution, (ii) 64 scans at a 4.0 ­cm−1 resolution; this characterization was to determine the identity of RO and LO biomass-associated performed at the Chemistry Analysis Service of our uni- microorganisms and (iii) to unravel their role in the HM versity (“Servei d’Anàlisi de Química”) (https://​sct.​uab.​cat/​ sorption process. Thus, to discuss the role of isolated micro- saq/​es) (UAB, Catalunya, Spain). The detailed procedures organisms in the HM sorption process, a multiple analyti- to determine the biomass pH, mineral composition, surface cal approach was followed by using different techniques: (i) 13 International Journal of Environmental Science and Technology area and surface charge are summarized in Supplementary 1 week at 27 °C using an Anaerocult system (Merck KGaA, Material (Text S1). Darmstadt, Germany). Antibiotic resistance profiles were recovered by applying an antibiotic susceptibility test based Characterization of microorganisms on the Kirby–Bauer disc diffusion method (Sayah et al. 2005). Here, the tested antibiotics and their respective con- Strain isolation Microbial strains were isolated from the centrations were as follows: ampicillin—10 μg, tetracy- RO and LO waste plants. For bacterial growth, 0.100 g of cline—30 μg, and nalidixic acid—30 μg. each plant was mixed with 10.0 mL of Luria–Bertani (LB) broth medium containing tryptone (10.0 g/L), yeast extract Genotypic characterization For genotypic characteriza- (5.00 g/L) and sodium chloride (10.0 g/L) at pH 7.0 and tion, the total genomic DNA was extracted from each iso- then placed in an orbital shaking incubator (Infors HT, Eco- late using a bacterial genomic DNA isolation kit (Norgen tron, Switzerland) at 150 rpm and 27 ± 1 °C for 24 h. Sub- Biotek Corp, Canada). The template DNA concentration sequently, 100 µL aliquots of each sample were spread onto was assessed by measuring the absorbance at 260 nm using LB agar plates consisting of LB broth medium along with an Ultrospec 1100 Pro UV/Vis spectrophotometer (Amer- bacteriological agar (15.0 g/L) and incubated again for 24 h sham Biosciences, USA). PCR cycling was executed with a at 27 ± 1 °C. Techne FTC 3G/02 thermal cycler (Techne, USA) and con- To determine the number of colony forming units per ducted for 5 min at 95 °C for initial denaturing, followed by volume (cfu/mL) in each original biomass (RO and LO), an amplification cycle, 23 cycles of 1 min at 95 °C, 1 min 0.1 mL of the corresponding biomass/solution suspension, at 55 °C, 2 min at 72 °C and a final extension of 10 min at which was 0.1 g of biomass in 10 mL of sterile double 72 °C. Amplicons were visualized by electrophoresis on a deionized water, was spread in triplicate onto LB agar plates. 1.2% agarose gel in 1 × TAE buffer (Figure S1) and further Then, the samples were incubated at 27 ± 1 °C for 24 h. purified with Monarch® nucleic acid purification kits (New England BioLabs, USA). DNA sequencing was performed Phenotypic characterization Phenotypic characteriza- at the UAB Genomics and Bioinformatics Service (“Servei tion of the different microorganisms associated with rice de Genòmica i Bioinformàtica”) (http://​sct.​uab.​cat/​genom​ and rapeseed biomass samples was performed from single ica-​bioin​forma​tica) (UAB, Catalunya, Spain). Universal colonies collected from well-isolated cultures grown on LB 16S rRNA forward 27F (AGA​GTT​TGG​ATC​MTGG​CTC​ plates. Morphological cell characteristics were examined AG) and reverse 1492R (GGT​TAC​CTT​GTT​ACG​ACT​T) with a conventional Olympus BH2 (Olympus, Japan) light primers were used. microscope. Gram, Wirtz–Conklin spore and capsule stain- To analyse the sequences of partial gene amplicons of 16S ing were performed according to Doestch (1981), Schaeffer rRNA and to exclude ambiguous data, SEQUENCHER soft- and Fulton (1933) and Anthony (1931), respectively. Oxi- ware version 5.4.6 (http://​www.​genec​odes.​com/) was used. dase activity was tested employing oxidase strips (Oxoid The obtained sequences were then compared to those exist- Ltd., Basingstoke, UK). Catalase enzymatic activity analy- ing in the NCBI database and matched with accessible refer- sis was performed by applying a 3% (v/v) ­H2O2 solution ences (www.n​ cbi.n​ lm.n​ ih.g​ ov) through the Ribosomal Data- on each isolate to evaluate enzyme-generated oxygen bub- base Project at Michigan State University (RDP-II) (http://​ ble formation (Bergey and Holt 1994). Moreover, DNase rdp.​cme.​msu.​edu/) and BLAST software (https://​blast.​ncbi.​ activity and the degradation of Tween 80 were examined nlm.​nih.​gov/​Blast.​cgi) (Cole et al. 2014). Finally, they were according to the methodology reported by Høvik Hansen deposited in the GenBank sequence database. and Sørheim (1991). The detection of phosphate-solubiliz- ing microorganisms was performed following the protocol Experimental procedures reported by Nath et al. (2017). Biochemical characterization was accomplished through BioMérieux™ Clinical Diagnos- Batch sorption experiments tic Kits (BioMérieux®sa, Marcy-l’Etoile, France). Further characterization was developed by analysing Sorption experiments were carried out at room tem- microorganism growth in different selective solid and/or perature (25 ± 1 °C). A multimetal solution composed of differential media after 24 h of incubation at 27 °C. These 0.18 mmol/L solutions of Cr(III), Cd(II), Cu(II) and Pb(II) media included Vogel–Bonner E (VBE; Southern Group was prepared. When conducting the isotherm sorption Laboratory, UK), minimal medium Vogel–Bonner (MMVB; experiments for single-metal systems, the initial heavy metal Southern Group Laboratory, UK), mannitol salt and Mac- concentrations ranged from 0.05 to 6 mmol/L. Conkey (Scharlau, Spain). In addition, haemophilic activity Batch sorption experiments were performed with the fol- was assessed through blood agar plates (Scharlau, Spain), lowing steps: 25 mg of each sorbent was placed in 5 mL and anaerobic growth was assayed on LB agar plates for Falcon tubes, and then, 2.5 mL of a heavy metal aqueous 13 International Journal of Environmental Science and Technology solution was added into the tube. Later, samples were placed Table 1  Comparison of the adsorption capacity of metal ions from in a rotary mixer (CE 2000 ABT-4, SBS Instruments SA, multiple metal systems (initial concentration of 0.18 mmol/L of Cr(III), Cu(II), Cd(II) and Pb(II)) by different adsorbents under the Barcelona, Spain) and shaken at 25 rpm for 24 h. The two same experimental conditions (T = 25 ± 1℃, 25 mg of adsorbent, phases were separated by decantation, and later, the aqueous 2.5 mL of solution and a contact time of 24 h) phase was filtered through 0.22 μm Millipore filters (Millex- Adsorbents Cr(III) Cu(II) Cd(II) Pb(II) GS, Millipore). Finally, inductively coupled plasma-mass q × ­103 q × ­103 q × ­103 q × ­103 spectrometry (ICP-MS, XSERIES 2 ICP-MS, Thermo Sci- (mmol/g) (mmol/g) (mmol/g) (mmol/g) entific, USA) was used to analyse the final concentration of heavy metals in the supernatant phase. Rice 9.81 ± 0.01 9.75 ± 0.01 10.5 ± 0.1 8.92 ± 0.01 The sorption/adsorption of the selected heavy metals by Rapeseed 7.24 ± 0.01 3.78 ± 0.01 6.76 ± 0.01 8.92 ± 0.01 the sorbents was expressed as the sorption percentage, which Pine 7.35 ± 0.01 4.02 ± 0.01 1.48 ± 0.01 9.09 ± 0.01 was calculated according to Eq. (1). Furthermore, the capac- Poplar 9.41 ± 0.01 7.04 ± 0.01 1.93 ± 0.01 7.04 ± 0.01 ity of the sorbent was calculated with Eq. (2) (Zhao et al. Corn 11.8 ± 0.1 10.1 ± 0.1 4.87 ± 0.01 1.78 ± 0.01 2019): Coffee shell 2.48 ± 0.01 4.61 ± 0.01 0.75 ± 0.01 20.1 ± 0.1 ( ) Sugar cane 2.46 ± 0.01 3.41 ± 0.01 0.52 ± 0.01 9.59 ± 0.01 C0 − Ce Cork 7.98 ± 0.01 5.92 ± 0.01 2.53 ± 0.01 11.5 ± 0.1 % Sorption∕Adsorption = × 100 (1) C0 Coffee grounds 3.48 ± 0.01 7.10 ± 0.01 0.14 ± 0.01 10.8 ± 0.1 ( ) C0 − Ce × V qe = (2) medium for 24 h at 27 °C was inoculated into 18.0 mL of LB m broth medium containing 0.18 mM of a heavy metal mixture where qe (mmol/g) is the sorption capacity of heavy metals, (Cr(III), Cd(II), Cu(II) and Pb(II)). For consortia, aliquots V (L) is the volume of the heavy metal solution, C0 and Ce of 0.5 mL of individual strain were inoculated according to are the initial and equilibrium heavy metal concentrations their isolation source into 18.0 (RO) or 18.5 (LO) mL of the in solution (both in mg/L), respectively, and m (g) is the above-mentioned LB mixed metal solution. Therefore, two dry weight of the sorbent. All the results are expressed as microbial consortia (corresponding to each biomass) were the mean value of minimum duplicate measurements, and formulated by mixing equal proportions of the pure cultures. the standard deviation (SD) was used to analyse data errors. Then, the cultures were placed in an orbital shaking incuba- Sorption experiments were carried out to optimize the tor (150 rpm) for 24 h at 27 °C. working conditions, including the influence of pH, initial After 24 h, the samples were centrifuged (Centrifuge metal concentration and contact time. Furthermore, kinet- 5430, Eppendorf, Germany) at 8000 rpm for 15 min. Finally, ics and isotherm models were chosen to study the sorption the solution was analysed by using ICP-MS (XSERIES 2 process. The pseudo-first-order (PFO), pseudo-second-order ICP-MS, Thermo Scientific, USA) to analyse the concentra- (PSO) and Elovich models (Wang et al. 2015) are typically tions of heavy metals in the supernatant phase. used to describe sorption processes, while the Langmuir and Freundlich isotherm models are typically used to describe the sorption mechanism for the interaction of heavy metal Results and discussion ions on a sorbent surface. The above-mentioned information details can be found in Supplementary Material (Text S2). Taking into account previous research with other biomass Additionally, experiments with sterilized biomasses were materials (Dai et al. 2018), different agricultural wastes are performed as described above. A complete autoclave cycle compared in Table 1 in terms of their sorption capacities (Raypa, Terrassa, Spain) was performed for 1 to 1.5 h at for the removal of HMs from multiple metal aqueous solu- 121 °C to eliminate live microorganisms, thereby allowing tions containing Cr(III), Cu(II), Cd and Pb(II). Pine, poplar, the uptake potential of sterile biomass to be tested. There- coffee shell, sugar cane and coffee grounds all show high fore, sorption experiments were prepared with common bio- sorption capacities for Cr(III), Cu(II) and Pb(II) and low masses and sterile biomasses, and the roles of the biomass- sorption capacities for Cd(II), as seen from the collected associated microorganisms in the metal uptake process were results (Table 1). In the case of corn wastes, it shows high evaluated. sorption capacities for Cr(III), Cu(II) and Cd(II) and low sorption capacities for Pb(II). It is worth noting that the pre- Exposure of isolated strains to a heavy metal solution pared RO and LO samples show good sorption capacities for all metal ions compared with the other sorbents listed here. For each individual microbial strain, an aliquot of 2.00 of In particular, the case of Cd(II) is highlighted because both each strain culture ­(OD600 = 1.2–1.4) grown on LB liquid 13 International Journal of Environmental Science and Technology Table 2  Total concentrations of the minerals and heavy metals (mg/Kg) along with the zeta potential and BET results of rice (RO) and rapeseed (LO) biomass Fe Mn Ni Cu As Pb Mg P K ZETA BET RO 6,291 2,348 70.5 747 15.2 1,691 2,051 25,108 28,700 −12.4 4.39 LO 6,410 208.1 40.5 229 15.5 568.7 554.0 24,280 37,900 −1.44 40.7 sorbents (RO and LO) show higher affinity for Cd than the majority of other biomass materials. In this work, the authors wanted to distinguish between the nonliving biomass and the living microorganism systems that are present in them. Nonliving biomass characterization The concentrations of some minerals (P, K and Mg) and major metals (Fe and Mn) in the rice (RO) and rapeseed (LO) biomass materials are listed in Table 2. RO and LO have similar concentrations of some minerals. However, they differ in the contents of some others, such as Mn and Mg, with RO having higher concentrations of these metal ions Fig. 1  ATR-FTIR spectra of RO and LO than LO; thus, RO has different physicochemical properties (Wang et al. 2015; Zhao and Zhou 2019). These differences have not been related to differences in the corresponding Microorganism identification treatment of crops but can have an understandable role in the sorption process of each biomass waste system. Phenotypic and genotypic characterization Table 2 also shows the zeta potential and BET values of RO and LO. Both biomass materials show negative zeta Through phenotypic analysis, a total of 8 microorganisms potential values, so a negative charge on their surface prob- associated with the rice (R01, R02, R03 and R04) and rape- ably facilitates the sorption of cation metal ions via electro- seed (L01, L02, L03 and L04) biomass samples were dif- static attraction (Plank and Christian 2007). In addition, the ferentiated. A summary of the isolated strain characteriza- difference in surface area of sorbents obtained from BET tion is shown in Table 3. After the colonies were grown on results gives enhanced surface area and a higher number of LB agar plates for 24 h at 27 °C, a preliminary phenotypic possible sorption sites for heavy metals (Sčíban et al. 2007) characterization of strains was performed, including macro- for LO in comparison with RO. scopic trait observations and cellular examination with opti- ATR-FTIR analysis was carried out to identify the func- cal microscopy. On the one hand, R01, R02, R02 and R04 tional groups present in the RO and LO biomass wastes that present a similar macroscopic appearance, forming smooth, might be involved in the biosorption process. In Fig. 1, the circular, nonpigmented and very mucoid colonies. On the FTIR spectra display several absorption peaks, indicating the other hand, L01 and L02 formed dry, nonpigmented, opaque complex nature of the examined biomaterials. As mentioned and mucoid colonies, while L03 formed small, raised and previously, cellulose, hemicellulose and lignin are the main white-coloured colonies, and L04 formed circular and con- components of agricultural straw biomass (Ghaffar and Fan vex colonies showing a bright yellowish pigment. Regard- 2014), and their functional groups (such as carboxylic and ing microscopic cellular features, R01, R02, R03 and R04 hydroxyl groups) can be found in the present biomasses (RO are short, rod-shaped and gram-positive; additionally, all of and LO) used as sorbents, which may participate in the pro- them, except R03, produce spores. In contrast, L01 and L02 posed metal sorption processes (Zhao et al. 2020). are rod-shaped, gram-negative and non-spore-forming bac- teria. L03 and L04 are gram-positive and non-spore-forming cocci. In the first strain, cells are organized in grape-like clusters, while they are arranged in tetrads in the second strain. They all showed different patterns of motility and capsule formation. To further characterize the isolated strains, a physiologic and metabolic analysis was developed. 13 International Journal of Environmental Science and Technology Table 3  Phenotypic and genotypic characterization of the microorganisms isolated from the rice and rapeseed biomass wastes Phenotypic characterization Characters RICE CANOLA Cell morphol- Rod-shaped Rod-shaped Rod-shaped Rod-shaped Rod-shaped Cocci in grape- Cocci arranged in ogy like clusters tetrads Gram stain + + + + − + + Spore formation + + − + − − − Motility + − − + + − − Capsule forma- − − − + + − − tion Enzymatic activity Catalase + + + + + + + Oxidase + + − − − − + H2S production − − − − − − − Urease − − − − − − − Indole produc- − − − − + − − tion DNase + − + + − + + Tween-80 + − − + + − − Phosphate 10 8 5 3 8 − − solubilization (mm) Antibiotic resistance Ampicilin S R R R R S R Tetracycline R R R S R R R Nalidixic Acid R R R R R R R Genotypic characterization Strain Bacillus sp. Bacillus sp. Bacillus sp. Bacillus sp. Escherichia coli Staphylococcus Micrococcus sp. R01 R02 R03 R04 L01 sp. L03 L04 Escherichia coli L02 Accession MK94285 MK934695 MK934698 MK937732 MK940239 MK942713 MK942714 number MK937745 All strains were positive for catalase, while only R01, R02 susceptibility patterns in regard to tetracycline and ampicil- and L04 were positive for oxidase. R01, R02, R03, R04, lin (Table 3). Last, rod-shaped strains from both the rice and L01 and L02 produce β-haemolysis, whereas L03 and L04 rapeseed biomasses present the ability to solubilize inor- induce an ⍺-haemolytic response. All isolates exhibited no ganic phosphorus from insoluble compounds. This capac- ­H2S production and were urease negative, and only L01 and ity, which is not detected with rapeseed coccoid strains, is L02 were positive for indole production. Moreover, all of considered one of the most relevant traits associated with them reveal distinct responses to DNA and Tween 80 (a non- plant phosphate nutrition by rhizosphere microorganisms ionic surfactant) hydrolysis (Table 3). R01, R02, R03 and (Alori et al. 2017). R04 are facultative anaerobes that develop a chemoorgano- For genotypic characterization, the DNA band of each heterotrophic metabolism, and L01, L02, L03 and L04 are strain obtained from PCR cycling was visualized in a 1.2% strictly aerobic bacteria. Regarding their growth on minimal, agarose gel before being further purified and sequenced selective and differential media, R01, R02, R03 and R04 (Supplementary Material, Figure S1). The processed were able to grow on mannitol salt agar and MMVB medium sequence was then entered into the Ribosomal Database Pro- but not on MacConkey agar or VBE medium. In the case of ject website at Michigan State University (RDP-II) (https://​ rapeseed-isolated microorganisms, L01 and L02 grew on rdp.​cme.​msu.​edu/​class​ifier/​class​ifier.​jsp). The sequence MacConkey agar and MMVB, while L03 and L04 grew only analysis of the isolated strains shows their genus with a on mannitol salt. The antibiogram results indicate that they 100% probability. The sequence was then introduced in are all resistant to nalidixic acid, but they show different BLAST (Basic Local Alignment Search Tool) (https://​blast.​ 13 International Journal of Environmental Science and Technology Fig. 2  Influence of the aqueous initial solution pH on the a RO and b LO sorption systems. Experimental conditions: T = 25 ± 1℃, 25 mg of sorbent, 2.5 mL of aqueous solution with 0.18 mmol/L of each element and a contact time of 24 h. The lines are a guide for the eyes ncbi.​nlm.​nih.​gov/​Blast.​cgi), displaying the following high As shown in Fig. 2, LO has a better sorption capacity than degrees of gene sequence similarity: R01 Bacillus thuring- RO, especially at pH ≥ 3.0. As expected, the metal sorption iensis (99.6%); R02 Bacillus subtilis (98.99%); R03 Bacillus percentage for both biomaterials increased with increasing pumilus (99.3%); R04 Bacillus cereus (99.8%); L01 and L02 pH. At pH 2.0, the lowest sorption percentages were found, Escherichia coli (99.71%); L03 Staphylococcus epidermidis probably because hydronium ions ­(H3O+) on the surface (99.79%); and L04 Micrococcus luteus (99.42%). According functional groups usually limit cation sorption due to their to these results, the isolates were identified only at the genus repulsive force (Feizi and Jalali 2015). Competitive sorption level since further gene sequences are needed to certainly of heavy metal cations is also found for both biomaterials, state that they belong to the specified species. At this point, as shown in Fig. 2. The sorption sequence of heavy metals the rapeseed-associated strain L01 was no longer considered by RO was ranked as Pb(II) > Cr(III) > Cd(II) > Cu(II) in all for future experiments, as it was estimated to be the same pH ranges, as shown in Fig. 2a. LO has a similar sorption as strain L02. trend. The higher adsorption percentage values found with In general, terms, these findings are understandable, tak- LO for all heavy metal cations can be related to its enhanced ing into account the origin of the samples, since the species surface area compared with RO (see Table 2), which leads to of Bacillus and Micrococcus genera are widely distributed possibly higher sorption sites available, as mentioned previ- in the environment (Maldonado et al. 2010; Hashem et al. ously. Considering that an aqueous solution pH above 5.0 2019). Moreover, isolated Bacillus strains have high phos- can cause heavy metal precipitation (Shen et al. 2017), pH phate solubilization potential, especially R01 and R02, 4.0 was chosen in the following studies. which is related to their rhizosphere (roots) provenance and their ability to improve plant nutrient uptake. On the other Kinetics modelling hand, Micrococcus has been extensively shown to be both phyllospheric and rhizospheric genera that promote plant The kinetics models used here to describe the sorption growth activities, specifically with rapeseed plants (Ghavami process include the PFO, PSO and Elovich models, which et al. 2017). The presence of E. coli suggests a faecal ori- can be described in Text S2 (Supplementary Material). gin and can be explained by possible contamination during The experimental kinetic data for RO and LO from Fig- sample transport and handling and/or the erratic distribution ure S2, following the corresponding procedure described of microorganisms in irrigation water (Luyt et al. 2012). In in Text S2, are adjusted to the models, as shown in Figure this way, the presence of Staphylococcus is presumably due S3. The RO and LO constant values from the kinetic to anthropogenic activities, since it is part of the human skin models, such as k i, q i, α and the calculated q e (experi- microbiome (Wood and Kelly 2010). mental sorption capacity), are listed in Table 4, together with the ­R 2 values of each model. Optimization of sorption conditions The results of RO and LO indicate that the PFO, PSO and Elovich equations all fit well with the experimental Influence of the solution pH data for all metal ions with high correlation coefficients (R2 > 0.960). In summary, the obtained results and model To check the influence of the pH of the aqueous metal solu- adjustments can explain why the biosorption processes of tion, the initial pH was adjusted to 2, 3, 4, 5 and 6 with RO and LO are rate-controlled mainly by physisorption, and either HCl or NaOH. The following detailed experimental they can also follow a multilayer biosorption process, which procedure is included in Text S2 (Supplementary Material). is probably related to the aromatic structure of lignin, which 13 International Journal of Environmental Science and Technology Table 4  Biosorption kinetic constant values for Cr(III), Cu(II), Cd(II) and Pb(II) by RO and LO for each model Experimental PFO PSO Elovich 3 3 2 3 2 qexp × ­10 k1 q1 × ­10 R k2 q2 × ­10 R A B R2 (mmol/g) (min−1) (mmol/g) (g/mmol*min) (mmol/g) Cr(III) RO 8.70 160 8.10 0.972 67.6 3.80 0.972 6,063 2.8*103 0.965 LO 9.90 0.624 9.70 0.995 322 9.80 0.997 7,152 3.1*1024 0.998 Cu(II) RO 3.80 0.107 3.70 0.961 67.6 3.80 0.971 6,063 2.8*103 0.968 LO 9.90 0.662 9.70 0.997 418 9.80 0.998 11,970 7.0*1044 0.998 Cd(II) RO 6.70 0.194 6.60 0.997 168 6.60 0.997 12,110 1.2*1028 0.997 LO 9.70 0.543 9.40 0.986 206 9.60 0.991 6,306 1.4*1020 0.992 Pb(II) RO 8.90 0.153 8.60 0.996 60.2 8.80 0.999 4,870 1.1*1012 0.998 LO 14.0 0.845 14.0 0.999 746 14.0 0.999 8,526 5.6*1044 0.999 Fig. 3  Freundlich and Langmuir biosorption isotherms of heavy metals in individual-element systems by a, b RO and c, d LO. Biosorption conditions: T = 25 ± 1 °C, pH 4.0, 25 mg of sorbent, 2.5 mL of aqueous solution and a contact time of 24 h. The lines are a guide for the eyes is predominant in the composition of both rice and rapeseed model assumes it occurs on a heterogeneous surface, which biomass wastes (Sharma et al. 2004; Hubbe 2021). can be described in Text S2 (in supplementary material). The plots of qe (sorption capacity) versus Ce (concentra- Isotherm modelling tion at the equilibrium state) of each metal in a system with a single heavy metal are shown in Fig. 3. The correspond- To evaluate the maximum biosorption capacity of RO and ing constant values are shown in Table S1, including the ­R2 LO, Langmuir and Freundlich isotherm models were used. values for each model (see Text S2 for further Langmuir Data results from the experiments of the initial metal con- constant explanation). centration variation (Figure S4) are used in this case (fol- In the present study, the Langmuir equation fits the exper- lowing the corresponding procedure described in Text S2). imental data better than the Freundlich equation in a system The Langmuir model assumes that the uptake of metal ions with a single heavy metal content (either by RO or LO). The occurs on a homogeneous surface, whereas the Freundlich Freundlich model cannot properly fit all the experimental 13 International Journal of Environmental Science and Technology Fig. 4  Comparison of the sorption capacities of non- sterile and sterile rice (RO) at different concentration levels of multimetal aqueous solu- tions. Experimental conditions: T = 25 ± 1 °C, pH 4.0, 25 mg of sorbent, 2.5 mL of metal solu- tion and a contact time of 24 h data, as the ­R2 values are generally lower than the R2 values live microorganisms to test the metal uptake of sterile bio- with the Langmuir model (shown in Table S1 and repre- mass. In the same way, the role of biomass-associated micro- sented in Fig. 3). In addition, the Langmuir RL values are organisms in the metal uptake process was quantified by between 0 and 1 for all metal ions in all systems and indicate using the original nonliving biomass. The sorption experi- that the biosorption of all the metal ions on RO and LO is ment results of both the original and sterile plant biomass favourable. The good agreement achieved with the Langmuir wastes are plotted in Figs. 4 and 5 for rice (RO) and rape- model suggests that there is probably an interaction between seed (LO), respectively. Solutions containing multiple heavy the metal species and homogeneous sorbent surfaces. metals in different amounts, from 0.05 to 4 mmol/L, were Furthermore, the maximum sorption capacity values for evaluated. Cr(III), Cu(II) and Pb(II) in systems with a single heavy Regarding RO, comparable findings were observed metal are properly collected in Table S1. As expected from (Fig. 4). A higher specific HM removal (q) with the original our previous results, RO shows lower maximum sorption biomass is clear for all the tested concentrations of Cr(III), capacities than LO for all heavy metals in this study, which Cu(II) and Cd(II), highlighting the removal obtained at ini- can be related to the different BET values of RO and LO tial concentrations in solution of approximately 4 mmol/L (Table 4). Compared with RO, LO has higher BET values, for Cr(III) and Cu(II). However, no bacterial resistance which corresponds to a higher surface; thus, higher possible to Pb(II) is observed here, indicating that the role of RO sorption sites and subsequently higher sorption rates are pos- microorganisms is insignificant and that most HM sorp- sible with LO, as evidenced here. tion is achieved by rice biomass, which is most efficient at 4 mmol/L. Comparison of nonliving sterile and nonsterile biomass The results concerning LO original biomass show higher sorption capacity sorption capacity removal values compared with micro- organism-free samples (sterile biomass), especially when To investigate the contribution of microorganisms to the HM exposed to Cr(III), Cu(II) and Pb(II) (Fig. 5). This behaviour sorption process, nonliving biomass with and without micro- can be explained by the complementary role of the associ- organisms was used as a sorbent for HM removal. ated microorganisms in the HM capture process. However, The RO and LO bacterial cfu counts in the original non- at 4 mmol/L Pb(II), a similar adsorption capacity (q) was living biomass were 4.3·101 and 3.7·101 cfu/mL, respec- detected between both systems (original biomass and sterile tively. Thus, the original biomass naturally contains the biomass, with and without microorganisms, respectively). associated microorganisms; consequently, rice and rapeseed Most likely, because microorganisms cannot survive at such biomass wastes were pretreated in an autoclave to eliminate high concentrations of Pb(II) (Yuan et al. 2015), they cannot 13 International Journal of Environmental Science and Technology Fig. 5  Comparison of the sorp- tion capacities of nonsterile and sterile rapeseed (LO) at different concentration levels of aqueous solutions with multiple HMs. Experimental conditions: T = 25 ± 1 °C, pH 4.0, 25 mg of sorbent, 2.5 mL of metal solu- tion and a contact time of 24 h have any role in the rice and rapeseed sorption capacity. Table 5  Sorption of HMs by microorganisms (mg/L). The initial HM Interestingly, microorganisms show a lower capacity for solution was 0.18 mmol/L for each element (Cr(III) 9.35, Cu(II) 11.4, Cd(II) 20.3 and Pb(II) 38.0 mg/L) Cd(II) removal than for the other metals under checked, presumably due to a low tolerance to this metal (Fig. 5c), as Strain Cr(III) Cu(II) Cd(II) Pb(II) reported previously (Villagrasa et al. 2021). CON- 0 0 0 0 These experiments demonstrated the likely role of bio- TROL* mass-associated microorganisms in enhancing heavy metal R01 0 0.180 ± 0.020 0 9.72 ± 0.08 removal (Ayangbenro and Babalola 2017), especially in the R02 0 0 0.700 ± 0.040 13.7 ± 0.1 case of rapeseed original nonliving biomass systems. Thus, R03 0 0 0 5.24 ± 0.08 to determine whether microorganisms exhibit resistance to R04 0 0 1.02 ± 0.04 14.8 ± 0.1 HMs and their corresponding adsorption capacity to remove L01–L02 0 0 0.620 ± 0.060 11.8 ± 0.1 HMs (at the same concentration range), the isolated micro- L03 0 0 0 4.64 ± 0.08 organisms were examined as sorbents in aqueous solutions L04 0 0 0 7.66 ± 0.08 with multiple HMs. Mix rice 8.66 ± 0.02 11.9 ± 0.1 18.4 ± 0.1 36.8 ± 0.1 strains Removal capabilities of the isolated microorganisms Mix canola 8.66 ± 0.02 11.9 ± 0.1 18.6 ± 0.2 37.1 ± 0.1 strains Metal exposure experiments with cell cultures were devel- oped to address the following: whether the strains can indeed contribute to HM removal and the amount of HM captured relatively high efficiencies, the most notable being 38.00% by them. These experiments were tested in a rich culture (strain R04), 35.30% (strain R02) and 30.44% (strains L01- medium (LB) to determine the role of microorganisms with- L02). The values collected for R01, R03, L03 and L04 are out being under nutrient starvation conditions. 25.02%, 13.50%, 11.95% and 19.72%, respectively. Nonethe- The results referred to this section are provided in Table 5. less, the involvement of the strains exposed to Cr(III), Cd(II) First, pure isolated microorganism cultures were evaluated. or Cu(II) can be dismissed. Although positive specific HM Once they reached the exponential phase of growth, they removal capacity (q) values are recovered for R01-Cu(II); were subjected to Cr(III), Cd(II), Cu(II) and Pb(II), each L01-Cd(II); L02-Cd(II); R02-Cd(II); and R04-Cd(II), they culture in separate aqueous solutions, at a final concentra- are too low to consider the strains as active participants tion of 0.18 mmol/L of each metal (in a multiple HM sys- in the HM uptake process. These findings may be largely tem). These experiments resulted in the active participation related to the metal toxicity susceptibility of these strains of all the isolated strains in the Pb(II) capture process, with when they are single cultured (Nguyen et al. 2015; Aparicio 13 International Journal of Environmental Science and Technology et al. 2018). It has been widely reported that certain con- et al. 2017; Jin et al. 2018). Moreover, it is well known that centrations of HM ions can affect the morphology, growth gram-positive microorganisms have excellent biosorption and metabolism of microorganisms by drastically altering capability due to their thick layer of peptidoglycan, which their cell membranes, changing their nucleic acid structure, includes an abundant number of sorption sites (Song and restricting enzyme activity, etc., which consequently reduces Tao 2013; Yin et al. 2019). cell viability (Fashola et al. 2016; Igiri et al. 2018). As RO and LO biomass microbiomes are composed of mixed populations, mixed cultures must also be explored. Conclusion The importance of testing consortia cultures remains in the cooperative actions they perform in an ecosystem that are In this work, rice (RO) and rapeseed (LO) residues are suc- not contemplated in pure cultures. As previously described, cessfully used as nonliving biomass sorbents for the removal consortia culture cells were grown to exponential phase and of Cr(III), Cu(II), Cd(II) and Pb(II) from multimetal aqueous then added to a 0.18 mmol/L HM multiple solution system. systems, and the sorption processes are investigated and dis- The results acquired here are remarkable, with some removal cussed in this paper. Furthermore, it is important to highlight capacity found in that case for Cr(III), Cd(II) and Cu(II), that this is the first time that the presence of microorganisms compared with previous results in single cultures. Particu- in these nonliving biomasses has been taken into account larly for Pb(II), as noted from the pure culture experiments and properly described, being different in each RO and LO (Table 5), both biomasses demonstrate the participation of case. Regarding the RO biomass, gram-positive bacteria are these consortia in the HM uptake process. The contribution exclusively from the genus Bacillus, while the LO contains percentage is over 80% of the total HM. Interestingly, similar both gram-positive bacteria that belongs to the genera Mic- ranges of metal removal and uptake efficiency (Pb(II) > Cd­( rococcus and Staphylococcus and gram-negative bacteria II) > Cu(II) > Cr(III)) were found with the consortia of both (genus Escherichia). The results show that both nonliving rice and rapeseed wastes. biomass residues can be used to remove heavy metals from wastewater systems, and the participation of isolated micro- Role of nonliving biomass‑associated organisms in consortia during the metal sorption process has microorganisms in the HM biosorption process been confirmed here. In this sense, it can be concluded that there is no need From the above-mentioned results, it can be confirmed that for any special pretreatment against the microorganisms in after 24 h of exposure, the microorganisms associated with the nonliving biomass waste samples of RO and LO prior to both biomasses play a role in the HM biosorption process. their use as metal sorbents, which implies their good feasi- In addition, with longer exposure times, these microorgan- bility for application from an economic point of view. isms (with some resistance to HM) would develop exponen- tially, and therefore, they would participate in promoting Supplementary Information The online version contains supplemen- tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 13762-0​ 22-0​ 4000-6. the HM biosorption process. Several studies have previously reported the use of a microbial consortium for remediation, Acknowledgements All the authors are grateful to L. López who which is considered more effective than single strain pure helped on the performance of some experiments and the analysis of culture (Dipak and Sankar 2015; Gupta and Kumar 2017). heavy metals by ICP-MS. As expected, microorganisms are more resistant to HMs in mixed cultures, probably due to the physiological diversity Authors’ contributions X.J. Shen, J. Zhao and N. Bonet-Garcia contrib- uted to conceptualization, methodology, formal analysis, investigation, between each strain and the different tolerance mechanisms data curation and writing—original draft preparation. E. Villagrasa was towards HMs. Therefore, this resistance will develop a prop- involved in conceptualization, methodology and writing—reviewing erly adapted and strong consortium through the exchange of and editing. A. Solé contributed to conceptualization, methodology genetic material between the present strains. Therefore, most and reviewing. X. Liao and C. Palet were involved in conceptualization, methodology, writing—reviewing, editing, supervision and funding strains can tolerate and remove these heavy metals and thus acquisition. All authors reviewed and approved the final manuscript assist rapeseed and rice biomass in HM elimination. for publication. Further studies are required to determine the mechanisms of the present strains on the HM removal capability and effi- Funding Open Access Funding provided by Universitat Auton- ciency and to understand the metabolic pathways and genes oma de Barcelona. This research was supported by grants from the National Key Research and Development Program of China (No. responsible for these processes (Mosa et al. 2016). How- 2018YFD0200904) and the Agricultural Science and Technology ever, it can be initially predicted that the obtained results Innovation Program of China (No. CAAS-ASTIP-2013-OCRI), by will be diverse since the genus Bacillus has been largely the Spanish research projects (Nos. CTM2015-65414-C2-1-R and described to adopt both metal biosorption and bioaccumula- AGL2015-70393-R) and by the China Scholarship Council (for J. Zhao—No. 201509110114). tion mechanisms (Çolak et al. 2011; Huang et al. 2018; Wu 13 International Journal of Environmental Science and Technology Data availability All data generated or analysed during this study are Brassica napus growth and amendment with chars from manure included in this manuscript. waste. Chemosphere 261:127798. https://d​ oi.o​ rg/1​ 0.1​ 016/j.c​ hemo​ sphere.​2020.​127798 Carolin CF, Kumar PS, Saravanan A, Joshiba GJ, Naushad M (2017) Declarations Efficient techniques for the removal of toxic heavy metals from aquatic environment: a review. J Environ Chem Eng 5(3):2782– Conflict of interset The authors declare no competing interests. 2799. https://​doi.​org/​10.​1016/j.​jece.​2017.​05.​029 Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Consent to publish The authors declare their consent for the publica- Porras-Alfaro A, Kuske CR, Tiedje JM (2014) Ribosomal data- tion of this manuscript. base project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 42(D1):633–642. https://​doi.​org/​10.​1093/​nar/​ Ethical approval Not applicable. gkt12​44 Çolak F, Atar N, Yazicioğlu D, Olgun A (2011) Biosorption of lead Consent to participate Not applicable. from aqueous solutions by Bacillus strains possessing heavy-metal resistance. 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