Biosorption potential of Purpureocillium lilacinum biomass for chromium (VI) removal: isolation, characterization, and significance of growth limiting factors

Abstract

Chromium (VI) is known to be harmful element that commonly found industrial waste, mining activities, and wastewater discharges from various industries. When released into the environment, Cr (VI) can contaminate soil, water, and air, posing a serious threat to living organisms. Aiming to Cr decontamination, this work was framed to isolate the fungal species having high Cr tolerance capacity and to exploit as bio-sorbent for the removal of Cr (VI) from aqueous solutions by biosorption. Among the fungal species isolated from the Cr (VI) contaminated soil sample, the filamentous fungus of Ophiocordycipitaceae family, Purpureocillium lilacinum was identified using molecular sequencing technique, showed maximum tolerance against Cr (VI) with a tolerance index of 1.19 ± 0.23. Further, Plackett Burman Design was applied to investigate for ascertaining the significance of different carbon and nitrogen sources on P. lilacinus growth, as well as the influence of environmental factors, such as pH, temperature, and Cr (VI) concentration. The results explicated that glucose was the most preferred carbon source for P. lilacinus, while yeast extract was the most preferred for nitrogen source. The optimum pH value and temperature were found to be 6.0 and 26 °C, respectively. In addition, P. lilacinus isolate was identified to survive in high concentrations of Cr (VI), indicating its potential for employing effective bioremediation of chromium-contaminated site.

Introduction

In recent decades, heavy mental pollution has been recognised as a serious threat to the environment due to rapid growth in industrialization and urbanization. In this context, there is a growing environmental and health concern towards revitalization of heavy metal polluted habitat including aquatic ecosystems regions [28]. Among the heavy metals, chrome may be found in both the trivalent [Cr (III)] and hexavalent [Cr (VI)] oxidation forms. Since Cr (VI) is categorized as more toxic, that can cause teratogenic effect to animals and human beings, however, Cr (III) is thought to be a significant trace element for mammalian metabolism [30]. Atomic number of the Cr is 24. Generally, it is found as transition metal oxides [Cr (II) to Cr (VI)]. Effluents from tanneries, fertilizers, pigment industries, sewage, oil well drilling, and fertilizer industries are the main sources of Cr. Based on the permeability property, Cr (VI) is considered to be quite hazardous than Cr (III). Cr (VI), on the other hand, actively traverses passageways seeking isoelectric and isostructural metal ions. Due to Cr phytotoxicity, crops are affected by inhibition of seed germination, leaf chlorosis, deficiency in root growth. Cr (VI) has been classified as a group 1 carcinogenic to humans, indicating the severity of the Cr (VI) hazard (Cancer.org, 2019). Consuming too much chromium can lead to liver and kidney damage, lowered immunity, respiratory troubles, and other health concerns [25].

In this light, it is crucial for removing the Cr (VI) from the environment or converted into the safer trivalent form. Chromium can be removed from the contaminated environment using different methods, such as ion exchange separation, membrane separation, chemical precipitation, filtering, and so on [8]. However, these approaches are resource-intensive, time-consuming, and technically challenging because they produce significant secondary wastes. In addition, it was shown that even low concentrations of heavy metals rendered these methods are found to be less-effective [17]. Consequently, the focus of cleaning efforts has lately shifted to the adsorption of heavy metals onto suitable adsorbents [37]. In the business world, activated carbon is utilised as an adsorbent to clean up the contaminated water supplies. However, commercial activated carbon is expensive because it required high production and regeneration cost [9] and lower capacity for adsorbing hexavalent chromium. This calls for a high-efficiency, low-cost adsorbent material. To address this issue, many studies have been carried out using biomass, such as fruit peels and shells but their main challenges was low production yield [16, 31].

As an alternative, the use of biological materials as adsorbents have been received a lot of interest in recent years (bio-sorbent). It is determined that the bio-sorbents are easily accessible, relatively inexpensive, and have a high adsorption effectiveness [3]. In this line, bacterial and fungal biomass, plant and animal solid waste, and synthetic biomaterials are the significant examples for contemporary use of bio-sorbents [23]. Among the aforementioned bio-sorbents, fungi provide significant advantages because they exhibit substantial metal binding capabilities, while comparing with other bio-sorbent materials in terms of biosorption and bioaccumulation efficiency [5, 6]. Fungi have attracted significant attention as bio-sorbent due to their high cell wall content, which suggests the presence of a diverse range of functional groups that can be involved in metal removal. When comparing the usage of fungi as bio-sorbents to that of algae and plants, it is easy to cultivate in large quantities of fungal biomass, which can be produced utilizing with simple processing techniques at low cost. In a recent study, the functional groups of Mucor rouxii involved in lead, chromium, cadmium, nickel, and zinc biosorption were studied [35]. It was proven from the study elsewhere, a chromium-resistant fungus isolate, Trichoderma sp. BSCR02, was collected from Manjakkudi lake and shown for efficient biosorption of chromium [24]. The biomass of Trichoderma sp. BSCR02, both in its natural state and after being treated with alkali, was tested for its capacity to biosorption of chromium. Furthermore, the biosorption can be improved by adjusting the pH, temperature, starting metal concentration, bio-sorbent concentration, and contact duration, all of which are operational limiting factors that influencing the biosorption [15]. Keeping in view of biosorption potential, the present work was aimed at investigating to isolate Cr tolerant fungal species as bio-sorbent for the removal of Cr (VI) from aqueous solution. Furthermore, the limiting parameters that crucially affect the biosorption were screened using Plackett Burman (PB) Design. In addition, the functional groups present in the fungal biomass which are responsible for Cr (VI) biosorption were investigate by FTIR [7].

Materials and methods

Isolation of Cr (VI) tolerant fungal species from contaminated soil sample

Figure 1 demonstrates the sematic flow of isolation of Cr tolerant fungi from polluted soil. The fungal species present in the collected soil sample were isolated using serial dilution and subsequent culturing on the Sabouraud dextrose agar or SDA medium (Glucose—40 g, Peptone—10 g, Agar—15 g, Distilled water—1000 mL and pH—5.6 ± 0.2) [33]. In such a way, 10 g of the collected soil sample was added to 100 mL of sterile distilled water in a 250 mL Erlenmeyer flask and the contents were shaken at 100 RPM for 15 min in a flask shaker to obtain a uniform suspension of soil. After through mixing, one mL of the suspension was transferred to 9 mL of sterile distilled water in a test tube and mixed well. This step was repeated for another four times under aseptic conditions to obtain 10⁻² to 10 ⁻⁵ dilutions. Following the serial dilution, 0.1 mL of the sample from each dilution were spread on the surface of SDA plates and then incubated at 27 °C for 7 days. During the period of incubation, fungal colonies with unique morphologies and microscopic characteristics (using lactophenol cotton blue staining) were picked out and cultured separately on SDA plates. After numerous repetitions, the isolated fungal strains were cultured on agar slants and then stored at 4 °C.

Fig. 1

The sematic flow of isolation of Cr tolerant fungi from polluted soil

Screening of chromium (VI) tolerant strains

The chromium (VI) tolerant strains from the isolated fungal strains were identified by culturing the fungal strains on SDA medium supplemented with K₂Cr₂O₇ (0.1 g/L) at 25 ^οC for 7 days [38]. The fungal growth was estimated by measuring the diameter of the colony (distance between the inoculation point and end of the longest hypha). To measure the diameter of the fungal colonies on chromium (VI) laden SDA medium, scans of the colonies were made using a digital camera and the images were analysed using image processing software ImageJ ver. 1.4 software [12]. Colonies with diameter greater than 4 cm were considered to have significant growth, therefore, notable tolerant to chromium (VI). In contrast, colonies with diameter lesser than 2 cm were considered to have no significant growth and hence, sensitive to chromium (VI) [38]. The capacity of the chromium (VI) tolerance was ascertained using the tolerance index (TI) factor of the fungal species that were grown in chromium (VI) laden medium. Tolerance index (TI) was measured using the Eq. (1).

$$TI=\frac{Fg(Cr)}{Fg}$$

(1)

where: TI: the tolerance index of fungi for Cr.

Fg (Cr): Fungal growth in chromium (VI) supplemented SDA medium.

Fg: Fungal growth in unsupplemented chromium (VI) SDA medium in the same time period

Identification of isolated fungal species tolerant to chromium (VI):

In order to identify the isolated fungal having highest TI capacity, the molecular identification technique was carried out using 18s rRNA analysis. Prior to analysis, the genomic DNA of the fungi was extracted and isolated using 0.8% agarose gel as per the standard protocol [13, 27]. After isolating the genomic DNA, PCR amplification of 18s rRNA gene was carried out using the universal forward (IT1SF) and reverse (IT1SR) primers. The purified PCR product was then subjected to Sanger’s sequencing and the obtained sequence was deposited into NCBI’s GeneBank database. Using NCBI’s BLASTn tool, a list of highly similar sequences and their identities were collected. The phylogeny between the collected and isolated chromium (VI) tolerant fungi sequence was inferred using MEGA ver. 11 software.

Characteristic analysis of soil sample used for microbial source

Selected chemical properties of soil sample were determined by the standard methods demonstrated by Association of official analytical chemists (AOAC). Elemental analysis (EDX) was carried out an instrument (Hitachi S-3400N, Japan). For increasing the image quality and electron conduction, dried soil sample was taken on to the sample mounted on aluminium stab sequenced which was coated by thin layer of gold under vacuum. Further, the elemental composition was deduced by an energy dispersive X-ray detector. An XRD instrument (Malvern Panalytical Ltd, UK) was employed for analysing the mineralogical components. XRD technique in combination with other synchrotron X-ray microanalytical analysis was used to confirm the geochemical forms of Cr, since the soil sample was collected from highly polluted site. Since Cr was identified as major toxic heavy metal contaminant, further the study was emphasised to analysis the toxic forms of Cr. Hence, XPS Analysis (PHI Genesis Instruments, US) was carried out for deducing the Cr (III), Cr (VI), and total Cr.

Ascertaining the growth medium and process conditions

Selection of appropriate carbon and nitrogen sources

The fungal isolate with maximum chromium (VI) tolerance was grown separately in the Sabouraud liquid medium containing 20 g/L different carbon sources, such as glucose, fructose, sucrose, maltose, and starch with the purpose of determining the effect of individual carbon source on the total fungal biomass yield. Likewise, the isolated fungus was grown in the Sabouraud liquid medium with 10 g/L of peptone, tryptone and yeast extract which tending to evaluate the influence of nitrogen source on the total fungal biomass yield. During the selection process, the environmental variables such as pH, temperature and K₂Cr₂O₇ concentration were kept constant at the fixed values, 5.6, 25 °C and 100 mg/L, respectively. Experiments were carried in the shake flasks using a shaking incubator with constant agitation at 150 RPM for 7 days.

Evaluation of process condition

Isolated fungal species was subjected to examine for determining the selected process factors for ameliorated growth performance. In such as a way, the isolate was cultured at various pH values (3–9), temperature (20–32 °C) and K₂Cr₂O₇ concentrations (0.05–0.25 g/L), while maintaining the concentrations of carbon and nitrogen source at 20 and 10 g/L of the medium, respectively, the effect of aforementioned environmental factors was studied.

Statistical optimization to single out the significant growth limiting factors using Plackett Burman design

The significance of growth liming factors including concentration of chromium (VI) in the medium were determined for the isolated Cr tolerant fungus using Plackett Burman Design (PBD). In this line, while designing the optimization experiments, the concentration of identified carbon and nitrogen source, K₂Cr₂O₇, K₂HPO₄, NaNO₃, MgSO₄·7H₂O, NaCl, pH, and temperature were chosen [1, 19]. In PBD, the chosen variables were analyzed at two levels, whereas; high and low levels were denoted by + 1 and − 1, respectively. The high and low levels of each chosen variable are listed in Table 1. In this sense, a total of 12 experimental runs with different combinations of selected variable were carried out (Table 2). After inoculation, the cultured flasks were maintained in a shaking incubator (at 150 RPM) for 7 days. Further, the yield of fungal biomass at each flask was determined and taken as a response for the PBD experimental analysis. The design and analysis for PBD experiments were executed using Minitab software (version 18, Minitab HK, China).

Table 1 The higher and lower levels of selected the variables applied for PBD analysis

Table 2 PBD for the evaluation of nine independent variables (in coded value) with the corresponding actual and predicted P. lilacinus biomass concentration

Results and discussion

Characteristics of contaminated soil used for microbe isolation

Table 3 provides selected chemical properties of contaminated soil sample. Scanning Electron Microscopy–Energy Dispersive X-ray analysis (SEM–EDX) was done to determine the elemental composition of the contaminated soil. As can be seen from Fig. 2 (EDX pattern), the components present in the soil sample were found to be Cr, O, C, Na, Ca, Au, Si, Fe, Cl, Mg, and Al. The EDX pattern of the soil sample indicated Cr was one of the major elements presented. As seen in Fig. 2, Cr counts in three places with high peaks were obtained. The Cr content in the soil sample was found to be 5.56 weight %, where the composition of other elements is given in Table 4. The results confirmed that the soil was significantly contaminated by Cr heavy metal. Further, the mineralogical components of the soil were analysed by XRD (Fig. 3). In this context, collected soil sample was characterized to identify the existing of minerals, however, quartz was found to be a chief mineral. There were also other minor minerals, such as zeolite, orthoclase, albite, tamarugite, microcline, and silicate. From the XRD results, it was found that the Cr was mainly bounded with manganese oxides and iron. While analysing the crystalline phases, magnesium chromate (VI) (MgCrO₄), chromium (III) vanadium oxide (CrVO₄), and potassium calcium chromium (III) fluoride (KCaCr₂F₉) were identified that were exhibited as strong peaks between 25 and 28°. To understand the information on toxicity of Cr, the oxidation states of the Cr elements were undertaken using XPS analysis (Fig. 4). The results showed that various chemical states of Cr occurred in the soil sample, including Cr (III), Cr (VI), and some other unknown states. Based on the height and area of the peaks, Cr (III) represented the highest proportion.

Table 3 Selected chemical properties of contaminated soil sample used for microbial isolation source

Fig. 2

SEM image and EDX spectrum of contaminated soil

Table 4 The wt.% that were observed from EDX analysis

Fig. 3

XRD spectrum of contaminated soil sample [whereas, Q-Quartz (SiO₂), T-Tamarugite, R-Rhodium Vanadium Oxide, O-Orthoclase, Z-Zeolite, Mi-Microcline (KAlSi₃O₈), A-Albite, MP-Manganese Phosphate)

Fig. 4

XPS spectrum for soil sample

Isolation and identification of Cr (VI) tolerant fungi

A total of 32 fungal colonies based on their colony morphology and microscopic features were isolated from different dilutions of the soil sample. Among these fungi, 5 fungi species were identified to have capable of growing in the SDA medium containing 0.1 g/L of K₂Cr₂O₇, which meant that these 5 fungi had ability for tolerating Cr (VI). Accordingly, the fungal isolates that showed tolerance against Cr (VI) were further designated as LT-1, LT-2, LT-3, LT-4 and LT-5 (Fig. 5). In order to compare the Cr (VI) tolerance among these fugus, tolerance index for of LT-1, LT-2, LT-3, LT-4 and LT-5 were determined, the same has been found to be 0.61 ± 0.10, 0.42 ± 0.17, 0.75 ± 0.09, 0.38 ± 0.12 and 1.19 ± 0.23, respectively. The study results on Cr TI revealed that that the isolate, designated as LT-5, had showed a high Cr TI compared to the other isolates. Hence, it was identified and selected as the potential strain for possible remediation of Cr (VI) bio-sorption studies. Furthermore, molecular identification technique was performed on the LT-5. In this respect, the agarose gel showing the genomic DNA and PCR product is depicted in Fig. 6a. Succeeded by, sanger sequencing was applied for the PCR product which showed the length of the sequence was found to be 652 bp. During BLASTn search, the sequence obtained showed 99% identity to isolate Purpureocillium lilacinum-SY45B-a (P. lilacinum) [Fig. 6b]. The partial 18S rRNA sequence was deposited at NCBI’s GenBank database and the accession identification number OP788028.1 has been granted https://www.ncbi.nlm.nih.gov/nuccore/OP788028.1. Purpureocillium lilacinum, previously known as Paecilomyces lilacinum, which is a filamentous fungus of Ophiocordycipitaceae family. It is widely present in diverse environment [10]. P. lilacinus is used in the field of agriculture as a biocontrol agent against parasitic nematodes of plant [5, 6]. However, in recent times, the heavy metal reduction capability of P. lilacinus, especially for the reduction/adsorption of hexavalent chromium has been also widely explored [4, 14, 34]. Aiming to assess the potential of isolated P. lilacinus growth and Cr (VI) bio-sorption, the medium composition and growth condition were further optimized.

Fig. 5

Different Cr tolerant fungi isolated from the contaminated soil

Fig. 6

Identification of fungal isolate. a Agarose gel image showing the ladder and PCR product and b phylogenetic tree of the fungal isolate

Effect of various carbon and nitrogen sources on P. lilacinus growth

The influence of different carbon and nitrogen sources on the production of P. lilacinus biomass is illustrated in Fig. 7. Among the different carbon sources, the highest concentration of biomass was seen in the glucose (12.25 ± 1.23 g/L), followed by fructose (11.82 ± 0.41 g/L), sucrose (8.51 ± 0.55 g/L) and maltose (8.14 ± 0.27 g/L). The lowest concentration of biomass (6.62 ± 0.84 g/mL) was observed when starch was used as the sole carbon source. Similarly, when using yeast extract as the sole nitrogen source, maximum biomass concentration of 12.72 ± 0.92 g/L was attained. The other nitrogen sources such as peptone and tryptone yielded the biomass concentration of 11.28 ± 0.65 and 10.26 ± 1.02 g/L, respectively. Hence it was apparent that the glucose was found to be most preferred carbon source for the isolated fungi, P. lilacinus. Numerous studies have observed an enhancement in spore production by P. lilacinus when glucose since it can be easily metabolised as a carbon source [21, 32]. Nitrogen is another important macro nutrient that is required for the growth of fungi and plays an important role in the regulation of secondary metabolism. Usually, fungi can able to utilize a wide variety of nitrogen sources present in the environment [26]. According to Wang et al., [29], the preferred nitrogen source for phosphate solubilization by P. lilacinus was soy bean powder. However, in the present study, the order of nitrogen source preference by the isolate, P. lilacinus, when exposed to Cr (VI) was obtained as follows: yeast extract > peptone > tryptone.

Fig. 7

Influence of various carbon and nitrogen sources on the growth of P. lilacinus

Effect of growth limiting factors for P. lilacinus

Effect of pH

pH is one of the important factors that can significantly influence the growth of the fungus. In case of soil dwelling fungus, the optimum pH was reported in the range of 5 to 9 and majority of fungi prefers slightly acidic condition for growth [18]. In the present study, the biomass of P. lilacinus concentration increased constantly with increasing pH and attained a maximum of 12.88 ± 1.15 g/L at pH of 6. Any further increase in pH has been found to lead the deterioration in the fungal growth. At the pH 9, the lowest biomass concentration of 7.45 ± 1.73 g/L was observed. The profile for biomass concentration of P. lilacinus isolate over different pH values is depicted in Fig. 8. A study by Sharma et al., 2011 showed that the heavy metal reduction ability of P. lilacinus was not severely affected by pH and the activity remains constant in both acidic and basic condition [20]. However, the results were supported by Xi-zhuo et al. for the optimum pH of P. lilacinus to fall between 6 and 7 [32].

Fig. 8

Influence of pH on the growth of P. lilacinus

Effect of temperature

The influence of temperature on the growth of P. lilacinus in presence of Cr (VI) is given in Fig. 9. From the figure, it can be observed from the results, while the temperature increased, the biomass concentration of P. lilacinus also found to be increased and reached the highest biomass concentration of 12.82 ± 0.66 g/L at 26 °C. Beyond 26 °C, the biomass concentration become saturated till 30 °C and started decline in growth after 30 °C. Thus, the optimum temperature for P. lilacinus isolate in presence of Cr (VI) was found to be 26 °C. P. lilacinus is known as mesophile and it is recognized to survive in the temperature range of 8 to 38 °C. However, the optimum temperature can be in the range from 25 to 30 °C [2]. In this study, it was found that the presence of Cr (VI) did not affected the optimum temperature of P. lilacinus.

Fig. 9

Influence of temperature on the growth of P. lilacinus

Effect of Cr (VI) concentration

P. lilacinus is one of the important soil fungi known to reduce hexavalent chromium at accelerated rate. Before using P. lilacinus for Cr (VI) bioremediation from contaminated soil, it is essential to understand the maximum Cr (VI) tolerant level of the isolated fungi. Hence, the growth of P. lilacinus in different concentration of K₂Cr₂O₇ was studied and it is illustrated in Fig. 10. It was found that the biomass concentration of P. lilacinus considerably declined with increasing concentration of K₂Cr₂O₇. At the lowest K₂Cr₂O₇ concentration of 0.05 g/L, the biomass concentration was determined as 12.9 ± 0.93 g/L. Meanwhile at the highest K₂Cr₂O₇ concentration of 0.25, the biomass concentration of 3.25 ± 0.18 g/L was observed. Hence, the ability of P. lilacinus to tolerate high concentration of hexavalent chromium suggested that the isolate can be used for bioremediation of chromium. Recently, numerous studies have stated the importance of chromium tolerant fungi for bioremediation purposes. For instance, a study by Gizaw et al., [11] have isolated 10 different species of yeast from vegetable farm soil that can tolerate chromium concentration up to 2500 ppm. Similarly, Singh et al., 2023 have proven that the fungal species isolated from different coal overburden strata (OBS) of Assam, India showed tolerance to multiple heavy metals like Cu²⁺, Cr⁶⁺, Ni²⁺ and Cd²⁺ up to 10 nM concentration of each heavy metal [22]. Fungal tolerance to Cr (VI) is facilitated by various biological processes such as biosorption, accumulation, transportation and redox reaction. In many cases of Cr (VI) tolerant fungi, reduction of toxic Cr (VI) to less toxic Cr (III) was observed and this microbial reduction is pivotal for bioremediation of Cr (VI) [36].

Fig. 10

Influence of Cr (VI) on the growth of P. lilacinus

Selection of significant variables using PBD

In the present study, Plackett Burman Design was implemented to evaluate the effect of high and low levels of various independent variables on the growth of P. lilacinus in presence of Cr (VI). A total of 9 independent variables were used for the design and 12 experiments with different combinations of independent variables were conducted. Table 2 lists the experimental runs with various combination of independent variable, and corresponding observed and predicted biomass concentration of P. lilacinus. The highest concentration of biomass was obtained when performing the experimental run number of 9, while lowest biomass concentration was seen in run number 5. The results of ANOVA (Analysis of Variance) performed over the experimental data were listed in Table 5. The independent variable with high F-value and low p-value in ANOVA was considered to have greatest effect on the biomass formation. Among all the studied independent variables, the concentration of K₂Cr₂O₇ had affected the growth of P. lilacinus significantly. Next to K₂Cr₂O₇, the concentration of glucose, yeast extract and pH affects the biomass formation to the most (p < 0.05). Other factors like K₂HPO₄, NaNO₃, MgSO₄•7H₂O, NaCl, and temperature showed very little effect over the growth of P. lilacinus. Figure 11 shows the standardized effect of various independent variable on P. lilacinus biomass formation. For the biomass concentration of P. lilacinus, the predicted R-squared value of 0.8446 was deduced to reasonable agreement with the Adjusted R-squared value of 0.9763. Similarly, the R-squared value of 0.9945 was in reasonable agreement with the adjusted R-square value. The adjusted R-squared value of 0.9763, which indicated that the model equation derived from PBD was found to be significant and could explain 97.63 percent of the variability in the biomass concentration data. Figure 12 depicts the residual plots of PBD model and the model equation in actual form is given below (Eq. (2)),

$$Biomass{ }\,concentration{ }\left( {g/L} \right)\, = \,3.268\, + \,0.07344{ }A\, + \,0.129{ }B{ }{-}{ }7.492{ }C\, + \,0.890{ }D{-\!\!-}0.082{ }E{-\!\!-}0.697{ }F{ }{-}{ }0.237{ }G\, + \,0.0892{ }H{ }{-}{ }0.01514{ }J$$

(2)

Where A, B, C, D, E, F and G are the concentration of glucose, yeast extract, K₂Cr₂O₇, K₂HPO₄, NaNO₃, MgSO₄ 7H₂O, and NaCl (g/L), H is the initial pH of the culture medium and J is the incubation temperature (^oC).

Table 5 Analysis of Variance (ANOVA) of the PBD

Fig. 11

The pareto chart showing the effect of nine chosen independent variables on the growth of P. lilacinus

Fig. 12

The residual plots from PBD

Figure 13 depicts the biomass obtained from submerged culture and filtrated culture (from run number 9 of PB design). It was used for Cr sorption studies. From FTIR spectra (range of 400–4000 cm⁻¹) obtained from heat inactivated biomass before and after and Cr (VI) adsorption has provided the information on the interactions of the probable cell–metal ions and presented in Figs. 14 and 15. From the analysis of biomass, shifted broad bands found at 3580, 1830 and 1400 cm⁻¹ while compared with Cr (VI) laden biomass which showed the approximately same absorption at 3620, 1890 and 1370 cm⁻¹. These were attributed to –NH groups, hydroxyl groups (–OH), carboxylate anions (COO-) and carbonyl groups (–CO). It was apparent that the decrease in peaks intensity of carboxylate ions peaks at 1615 and 1418, 1280 cm⁻¹ which explicated of SO₃ groups and others absorption peaks at 1155 and 973 cm⁻¹ were attributed to the phosphate group (P–OH and P=O and stretching). Such results demonstrated that these functional groups were significantly contributed in the biosorption process. The peaks at lower wavenumbers (below 700 cm⁻¹) have also observed when comparison with dried Cr unloaded biomass. This might be attributed to an interaction N-containing bioligands and Cr (VI) species. EDAX spectra found before and after Cr (VI) biosorption onto heat inactivated biomass are illustrated in Fig. 16a and b, respectively. These spectra demonstrated very clearly the presence Cr (VI) in heat inactivated biomass. This analysis confirmed the significant amount of Cr (VI) absorption which revealed with Cr (VI) signals.

Fig. 13

Biomass for Cr (VI) biosorption (a) submerged culture and b filtrated culture (from run number 9 of PB design)

Fig. 14

IR spectra of heat inactivated biomass before Cr (VI) biosorption

Fig. 15

IR spectra of heat inactivated biomass after Cr (VI) biosorption

Fig. 16

EDAX spectra of biomass inactivated by heat (a) before absorption (b) after absorption