Do microorganisms enhance Fe (II) oxidation in the Nottingham biofiltration system at circumneutral pH?
2. Materials and methods
2.1 Bacterial strains used for Fe (II) oxidation tests
Acinetobacter sp. LB1 and an isolated strain of Burkholderia sp. strain LB2 (identity confirmed via API20 NE analysis (see supplementary data) were previously isolated from a biofiltration system treating borehole water and routinely maintained and cultivated in MSVP medium (minimal salts medium containing vitamins and pyruvate) as reported by Beukes and Schmidt (2012). Leptothrix mobilis (DSM 10617) was grown and maintained in a medium suggested by Spring et al. (1996) and Sphaerotilus natans (DSM 565) (Stokes, 1954) was grown and maintained using beef extract agar as suggested by the supplier’s instructions.
Sphaerotilus natans was inoculated into tap water covered beef extract agar slants and incubated between 20-25°C for at least 48 hours before further use in experiments. Stock cultures of the aforementioned strains were kept at -80°C in their respective culture media in the presence of 20% glycerol.
2.2 Fe (II) toxicity tests
The impact of varying concentrations of Fe (II) on the growth of two selected proteobacterial strains was used to establish the minimum Fe (II) concentration potentially inhibiting microbial growth in the biofiltration system. E. coli (ATCC 8739) was used as a well-known non-iron oxidizing reference strain representing the proteobacterial group of microorganisms.
80 Acinetobacter sp. LB1 was used to determine the toxicity of Fe (II) on an organism isolated from the biofiltration system. The assay was carried out in LBB (Leucoberbelin blue) medium suggested by Krumbein and Altmann (1973) and modified according to Beukes and Schmidt (2012). The nominal concentrations of Fe (II) used were: 0, 2, 4, 6, 8 and 10 mg/L.
Erlenmeyer flasks (100 mL) containing 30 mL of the above medium were inoculated with 3×105 cells (pregrown in LBB medium) per mL and incubated in a shaker incubator (MRC orbital shaker incubator, Israel) at 25°Cand 150 rpm. To monitor cell growth, 1 mL samples from each flask were sampled at 2hr intervals and the density of biomass formed was measured at OD600. One milliliter of sterile LBB medium was used as a blank.
2.3 Ferrozine based Fe (II) quantification
A ferrozine assay suggested by To et al. (1999) was used to test for the presence of Fe (II) and total iron within samples. The assay was carried out in 25 mL volumetric flasks.
Reagents used for the assay were as follows: ferrozine [4.9 mM - stock solution in distilled water], Fe (II) supplied in the form of ammonium ferrous sulfate [100 mg/L in acidified (6M HCl, pH 2) distilled water for the standard samples - stock solution], hydroxylamine hydrochloride [10% w/v - stock solution in distilled water] and ammonium acetate buffer (pH 7-7.5) [30% ammonium hydroxide - 467 mL, glacial acetic acid - 230 mL, made up to 1L with distilled water - stock solution]. The presence of Fe (II) was detected by a violet color resulting from the interaction of Fe (II) ions and ferrozine. Two standard curves were prepared, one employing hydroxylamine hydrochloride and the other without. The addition of hydroxylamine hydrochloride served to reduce Fe (III) to Fe (II), in order to measure the total iron in the system. Fe (III) values were obtained by subtracting Fe (II) from the total iron. For the standard curve, standard Fe (II) samples were prepared from the ammonium ferrous sulfate stock solution as follows: 0, 2, 4, 6, 8 and 10 mg/L Fe (II).
Analysis of experimental and standard samples for Fe (II) without the addition of hydroxylamine hydrochloride included an initial centrifugation step (3 minutes, 25°C, 14000 g) to remove any Fe (III) formed in the flasks. Samples with hydroxylamine hydrochloride for total Fe (II) measurements were measured directly. The Fe (II) standard and experimental samples were prepared by adding 0.5 mL ferrozine reagent, 20 mL of the respective Fe (II) standard (diluted appropriately using acidified distilled water, pH 2) or experimental sample,
81 0.5 mL hydroxylamine hydrochloride (when measuring total iron), 1.25 mL ammonium acetate buffer and the volume was made up to 25 mL in a volumetric flask with distilled water. The samples were thoroughly mixed and incubated in the dark at room temperature for 15 minutes to allow for color development. For the spectrophotometric quantification of Fe (II) in the system, the absorbance of a 2 mL sample was measured at 562 nm using a BIO- RAD Smartspec™ Plus. A sample without the addition of iron but only 20 mL of distilled water was used as a blank.
2.4 Comparison of biological and abiotic Fe (II) oxidation rates
Acinetobacter sp. LB1 was employed for the biological Fe (II) oxidation test with the intention to determine whether this Mn (II) oxidizing isolate has the ability to oxidize Fe (II) and to determine the potential contribution of microorganisms to Fe (II) oxidation at neutral pH under aerobic conditions. Iron oxidation tests were carried out in separate sets of 100 mL Erlenmeyer flasks containing 30 mL of 0.85% saline at pH 7 with additional controls run at pH 2.42 (acidified with HCl) and at pH 8.76 (5 mM HEPES buffer). Tests were conducted in the absence of bacteria (abiotic), in the presence of resting cells (Acinetobacter sp. LB1) (biological), in the presence of heat inactivated bacterial cells (Acinetobacter sp. LB1, 121°C for 15 minutes) or bacterial cells poisoned with 3.5% v/v formaldehyde. Resting cells were prepared by growing an overnight culture of Acinetobacter sp. LB1 in LBB medium and then washing and resuspending the cells in 0.85% saline. The tests were conducted over a 120 minute period for the test conducted at neutral pH and for 60 minutes for the low and high pH tests. The samples were incubated in a shaker incubator at 25°C and 250 rpm (MRC orbital shaker incubator, Israel). Flasks used for the test conducted at neutral pH were spiked with 4.50 mg/L Fe (II) and 5.70 mg/L for the low and high pH tests. Flasks containing bacterial cells were inoculated with 1×108 cells/mL. The ferrozine assay was employed to measure Fe (II) concentrations in samples before and after incubation, using the sample analysis procedure outlined in 2.3.
82 2.5 Fe (II) tests simulating biofilter conditions
This test was divided into two parts, quantitative and qualitative analysis of Fe (II) oxidation.
For the qualitative analysis, flasks (500 mL) containing borehole water (100 mL) and filter sand (10 g) (manganese greensands - glauconite with manganese oxides of various Mn valence states - intended to specifically remove Mn (II), Fe (II), hydrogen sulfide, and arsenic (Casale et al., 2002)) were set up. The sand granules were washed three times with distilled water and autoclaved at 121°C for 15 minutes prior to experimental analysis. Both control and experimental flasks were set up. Separate sets of flasks (500 mL Erlenmeyer flasks) were set up, employing borehole water (100 mL) and filter sand, spiked with the previously isolated Acinetobacter sp. LB1, an additional proteobacterial isolate from the biofiltration system - Burkholderia sp. strain LB2 (isolated in the same manner as Acinetobacter sp. LB1 as specified in Beukes and Schmidt (2012)) and two known manganese and iron oxidizing reference strains, Leptothrix mobilis (DSM 10617) and Sphaerotilus natans (DSM 565).
Flasks containing 100 mL of borehole water and filter sand were spiked with 1×103 cells/mL of the respective bacterial strains to determine whether the addition of these microorganisms had any effect on the oxidation of Fe (II) in these flasks. A control containing borehole water and filter sand with native microorganisms (7.04×105 cfu/mL for the iron oxidizers, iron oxidizing bacteria were quantified in a similar manner to MOB although the MSVP (minimal salts vitamins pyruvate) medium used to quantify the iron oxidizers contained 2 mg/L iron sulfate instead of manganese sulfate (Beukes and Schmidt, 2012)), without the addition of any other microorganisms was also established.
Samples were incubated in a shaker incubator (MRC orbital shaker incubator) at 25°C and 150 rpm for 7 days. As particulate matter from the filter sand affected the detection of Fe (II) in the system, only flasks without the addition of filter sand were analyzed using the ferrozine assay (2.3) to determine the amount of Fe (II) present in samples. Flasks with filter sand were therefore only used to visually demonstrate the effects of Fe (II) oxidation in the flasks, simulating conditions in the biofiltration system. The flask spiked with Acinetobacter sp. LB1 was checked for the presence of Fe (II) oxidation products formed over the 7-day period, using an Oxford (X-MAX) energy dispersive X-ray (EDX) detector coupled to a scanning electron microscope (Zeiss Evo LS 15, Germany). For the quantitative analysis, batch tests were conducted using freshly collected borehole water (pH 7) from the borehole water tank at
83 the biofiltration system to somewhat simulate the biofilter conditions. This test was used to determine the difference in Fe (II) oxidation rates in the presence of native microorganisms and in the presence of iron oxidizing reference strains. The same sets of experimental and control flasks were set up like in the qualitative analysis except only borehole water was used in the quantitative analysis.
2.6 Chemicals
Ferrozine and hydroxylamine hydrochloride were obtained from Sigma-Aldrich (South Africa). Unless otherwise stated all other chemicals used were of the highest purity commercially available.