Integrating bioelectrochemical system with aerobic bioreactor for organics removal and caustic recovery from alkaline saline wastewater.

Bioelectrochemical systems (BES) are increasingly being explored as an auxiliary unit process to enhance conventional waste treatment processes. This study proposed and validated the application of a dual-chamber bioelectrochemical cell as an add-on unit for an aerobic bioreactor to facilitate reagent-free pH-correction, organics removal and caustic recovery from an alkaline and saline wastewater. The process was continuously fed (hydraulic retention time (HRT) of 6 h) with a saline (25 g NaCl/L) and alkaline (pH 13) influent containing oxalate (25 mM) and acetate (25 mM) as the target organic impurities present in alumina refinery wastewater. Results suggested that the BES concurrently removed the majority of the influent organics and reduced the pH to a suitable range (9-9.5) for the aerobic bioreactor to further remove the residual organics. Compared to the aerobic bioreactor, the BES enabled a faster removal of oxalate (242 ± 27 vs. 100 ± 9.5 mg/L.h), whereas similar removal rates (93 ± 16 vs. 114 ± 23 mg/L.h, respectively) were recorded for acetate. Increasing catholyte HRT from 6 to 24 h increased the caustic strength from 0.22% to 0.86%. The BES enabled caustic production at an electrical energy demand of 0.47 kWh/kg-caustic, which is a fraction (22%) of the electrical energy requirement for caustic production using conventional chlor-alkali processes. The proposed application of BES holds promise to improve environmental sustainability of industries in managing organic impurities in alkaline and saline waste streams.

Effect of ferrous iron loading on dewaterability, heavy metal removal and bacterial community of digested sludge by Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans ILS-2 was adapted in digested sludge and used to treat sludge for dewaterability improvement. Results showed that increasing ferrous iron loading increased sludge dewaterability, but the inoculation of the bioleaching strain had little effect on sludge dewaterability compared to controls without the strain. The total extracellular polymeric substances (EPS) contents of sludges with and without bioleaching treatment were similar except for bioleaching treatment at 10% ferrous iron loading (on sludge total solids) where total EPS was higher with bioleaching treatment. However, bioleaching treatment for 48 h had a notable effect on removal of heavy metals, such as Mn, Ni and Zn, especially at the high loadings of ferrous iron. In the presence of A. ferrooxidans, the removal of Ni, Mn and Zn reached 93%, 88% and 80%, respectively, at a ferrous iron loading of 21%. The sequencing of 16S rRNA genes indicated that increasing ferrous iron loadings to 15% and 21% increased the relative abundance of Acidithiobacillus, Acidocella (with A. ferrooxidans) and Carboxylicivirga (without A. ferrooxidans) but decreased the abundance of Pseudomonas and Acinetobacter after 48 h treatment. This study enhanced the understanding of the correlations between bioleaching treatment of digested sludge, sludge dewaterability, heavy metal removal and bacterial communities.

Genome-based classification of Acidihalobacter prosperus F5 (=DSM 105917=JCM 32255) as Acidihalobacter yilgarnensis sp. nov.

The genus Acidihalobacter has three validated species, Acidihalobacter ferrooxydans, Acidihalobacter prosperus and Acidihalobacter aeolinanus, all of which were isolated from Vulcano island, Italy. They are obligately chemolithotrophic, aerobic, acidophilic and halophilic in nature and use either ferrous iron or reduced sulphur as electron donors. Recently, a novel strain was isolated from an acidic, saline drain in the Yilgarn region of Western Australia. Strain F5T has an absolute requirement for sodium chloride (>5 mM) and is osmophilic, growing in elevated concentrations (>1 M) of magnesium sulphate. A defining feature of its physiology is its ability to catalyse the oxidative dissolution of the most abundant copper mineral, chalcopyrite, suggesting a potential role in biomining. Originally categorized as a strain of A. prosperus, 16S rRNA gene phylogeny and multiprotein phylogenies derived from clusters of orthologous proteins (COGS) of ribosomal protein families and universal protein families unambiguously demonstrate that strain F5T forms a well-supported separate branch as a sister clade to A. prosperus and is clearly distinguishable from A. ferrooxydans DSM 14175T and A. aeolinanus DSM14174T. Results of comparisons between strain F5T and the other Acidihalobacter species, using genome-based average nucleotide identity, average amino acid identity, correlation indices of tetra-nucleotide signatures (Tetra) and genome-to-genome distance (digital DNA-DNA hybridization), support the contention that strain F5T represents a novel species of the genus Acidihalobacter. It is proposed that strain F5T should be formally reclassified as Acidihalobacter yilgarnenesis F5T (=DSM 105917T=JCM 32255T).

Biogenic Iron Sulfide Nanoparticles to Enable Extracellular Electron Uptake in Sulfate-Reducing Bacteria.

Microbes synthesize cell-associated nanoparticles (NPs) and utilize their physicochemical properties to produce energy under unfavorable metabolic conditions. Iron sulfide (FeS) NPs are ubiquitous and are predominantly biosynthesized by sulfate-reducing bacteria (SRB). However, the biological role of FeS NPs in SRB remains understudied. Now, conductive FeS NPs function is demonstrated as an electron conduit enabling Desulfovibrio vulgaris Hildenborough, an SRB strain, to utilize solid-state electron donors via direct electron uptake. After forming FeS NPs on the cell surface, D. vulgaris initiated current generation coupled with sulfate reduction on electrodes poised at -0.4 V versus standard hydrogen electrode. Single-cell activity analysis showed that the electron uptake and metabolic rate via FeS NPs in D. vulgaris were about sevenfold higher than those via native cell-surface proteins in other SRB.

Genome-based classification of two halotolerant extreme acidophiles, Acidihalobacter prosperus V6 (=DSM 14174 =JCM 32253) and 'Acidihalobacter ferrooxidans' V8 (=DSM 14175 =JCM 32254) as two new species, Acidihalobacter aeolianus sp. nov. and Acidihalobacter ferrooxydans sp. nov., respectively.

Phylogenomic analysis of recently released high-quality draft genome sequences of the halotolerant acidophiles, Acidihalobacter prosperus V6 (=DSM 14174=JCM 32253) and 'Acidihalobacter ferrooxidans' V8 (=DSM 14175=JCM 32254), was undertaken in order to clarify their taxonomic relationship. Sequence based phylogenomic approaches included 16S rRNA gene phylogeny, multi-gene phylogeny from the concatenated alignment of nine selected housekeeping genes and multiprotein phylogeny using clusters of orthologous groups of proteins from ribosomal protein families as well as those from complete sets of markers based on concatenated alignments of universal protein families. Non-sequence based approaches for species circumscription were based on analyses of average nucleotide identity, which was further reinforced by the correlation indices of tetra-nucleotide signatures as well as genome-to-genome distance (digital DNA-DNA hybridization) calculations. The different approaches undertaken in this study for species tree reconstruction resulted in a tree that was phylogenetically congruent, revealing that both micro-organisms are members of separate species of the genus Acidihalobacter. In accordance, it is proposed that A. prosperus V6T (=DSM 14174 T=JCM 32253 T) be formally classified as Acidihalobacter aeolianus sp. nov., and 'Acidihalobacter ferrooxidans' V8T (=DSM 14175 T=JCM 32254 T) as Acidihalobacter ferrooxydans sp. nov., and that both represent the type strains of their respective species.

Role of microorganisms in bioleaching of rare earth elements from primary and secondary resources.

In an era of environmental degradation, and water, and mineral scarcity, enhancing microbial function in sustainable mining has become a prerequisite for the future of the green economy. In recent years, the extensive use of rare earth elements (REEs) in green and smart technologies has led to an increase in the focus on recovery and separation of REEs from ore matrices. However, the recovery of REEs using traditional methods is complex and energy intensive, leading to the requirement to develop processes which are more economically feasible and environmentally friendly. The use of phosphate solubilizing microorganisms for bioleaching of REEs provides a biotechnical approach for the recovery of REEs from primary and secondary sources. However, managing and understanding the microbial-mineral interactions in order to develop a successful method for bioleaching of REEs still remains a major challenge. This review focuses on the use of microbes for the bioleaching of REEs and highlights the importance of genomic studies in order to narrow down potential microorganisms for the optimal extraction of REEs.

Biodegradability of legacy crude oil contamination in Gulf War damaged groundwater wells in Northern Kuwait.

During the 1991 Gulf War, oil wells in the oil fields of Kuwait were set aflame and destroyed. This resulted in severe crude oil pollution of the countries only fresh water aquifers. Here, for the first time the natural attenuation and biodegradation of the persisting groundwater contamination was investigated to assess potential processes in the aquifer. Biodegradation experiments were conducted under aerobic and multiple anaerobic conditions using microcosms of the contaminated groundwater from Kuwait. Under the conditions tested, a portion of the total petroleum hydrocarbon (TPH) component was degraded, however there was only a slight change in the bulk concentration of the contaminant measured as dissolved organic carbon (DOC), suggesting the presence of a recalcitrant pollutant. Changes in the associated microbial community composition under different reduction-oxidation conditions were observed and known hydrocarbon degraders identified. The results of this study indicate that lingering contaminant still persists in the groundwater and is recalcitrant to further biodegradation, which presents challenges for future remediation plans.

Competition between Naegleria fowleri and Free Living Amoeba Colonizing Laboratory Scale and Operational Drinking Water Distribution Systems.

Free living amoebae (FLA), including pathogenic Naegleria fowleri, can colonize and grow within pipe wall biofilms of drinking water distribution systems (DWDSs). Studies on the interactions between various FLA species in biofilms are limited. Understanding the interaction between FLA and the broader biofilm ecology could help better predict DWDS susceptibility to N. fowleri colonization. The aim of this study was to determine if N. fowleri and other FLAs ( Naegleria, Vermamoeba, Willaertia, and Vahlkampfia spp.) cocolonize DWDS biofilm. FLAs commonly isolated from DWDSs ( N. fowleri, V. vermiformis, and N. lovaniensis) were introduced into laboratory-scale biomonitors to determine the impact of these amoebae on N. fowleri's presence and viability. Over 18 months, a single viable amoebae ( N. fowleri, N. lovaniensis, or V. vermiformis) was detected in each biofilm sample, with the exception of N. lovaniensis and N. fowleri, which briefly cocolonized biofilm following their coinoculation. The analysis of biofilm and bulk water samples from operational DWDSs revealed a similar lack of cocolonization with a single FLA detected in 99% ( n = 242) of samples. Interestingly, various Naegleria spp. did colonize the same DWDS locations but at different times. This knowledge furthers the understanding of ecological factors which enable N. fowleri to colonize and survive within operational DWDSs and could aid water utilities to control its occurrence.

Identifying and Quantifying the Intermediate Processes during Nitrate-Dependent Iron(II) Oxidation.

Microbially driven nitrate-dependent iron (Fe) oxidation (NDFO) in subsurface environments has been intensively studied. However, the extent to which Fe(II) oxidation is biologically catalyzed remains unclear because no neutrophilic iron-oxidizing and nitrate reducing autotroph has been isolated to confirm the existence of an enzymatic pathway. While mixotrophic NDFO bacteria have been isolated, understanding the process is complicated by simultaneous abiotic oxidation due to nitrite produced during denitrification. In this study, the relative contributions of biotic and abiotic processes during NDFO were quantified through the compilation and model-based interpretation of previously published experimental data. The kinetics of chemical denitrification by Fe(II) (chemodenitrification) were assessed, and compelling evidence was found for the importance of organic ligands, specifically exopolymeric substances secreted by bacteria, in enhancing abiotic oxidation of Fe(II). However, nitrite alone could not explain the observed magnitude of Fe(II) oxidation, with 60-75% of overall Fe(II) oxidation attributed to an enzymatic pathway for investigated strains: Acidovorax ( A.) strain BoFeN1, 2AN, A. ebreus strain TPSY, Paracoccus denitrificans Pd 1222, and Pseudogulbenkiania sp. strain 2002. By rigorously quantifying the intermediate processes, this study eliminated the potential for abiotic Fe(II) oxidation to be exclusively responsible for NDFO and verified the key contribution from an additional, biological Fe(II) oxidation process catalyzed by NDFO bacteria.

Improvement of carbon usage for phosphorus recovery in EBPR-r and the shift in microbial community.

Enhanced biological phosphorus removal and recovery (EBPR-r) is a biofilm process that makes use of polyphosphate accumulating organisms (PAOs) to remove and recover phosphorus (P) from wastewater. The original process was inefficient, as indicated by the low P-release to carbon (C)-uptake (Prel/Cupt) molar ratio of the biofilm. This study successfully validated a strategy to improve the Prel/Cupt ratio by at least 3-fold. With an unchanged supply of carbon in the recovery stream, an increase in the hydraulic loading in stages I, II and III (7.2, 14.4 and 21.6 L, respectively) resulted in a 43% increase in the Prel/Cupt ratio (0.069, 0.076 and 0.103, respectively). The ratio further increased by 150% (from 0.103 to 0.255) when the duration of the P uptake period was increased from 4 h (stage III) to 10 h (stage IV). Canonical correspondence analysis showed that, correlated to the 3-fold increase in the Prel/Cupt ratio, there was an increase in the abundance of PAOs ("Candidatus Accumulibacter" Clade IIA) and a decrease in the occurrence of glycogen accumulating organisms (GAOs) (family Sinobacteraceae). However, the four stage operation impaired denitrification, resulting in a 5-fold reduction in the Nden/Pupt ratio. The decline in denitrification was consistent with a decrease in the abundance of denitrifiers including denitrifying PAOs (family Comamonadaceae and "Candidatus Accumulibacter" Clade IA). Overall, a strategy to facilitate more efficient use of carbon was validated, enabling a 3-fold carbon saving for P recovery. The new process enabled up to 80% of the wastewater P to be captured in a P-enriched stream (>90 mg/L) with a single uptake/release cycle of recovery.

Characterization of a Drinking Water Distribution Pipeline Terminally Colonized by Naegleria fowleri.

Free-living amoebae, such as Naegleria fowleri, Acanthamoeba spp., and Vermamoeba spp., have been identified as organisms of concern due to their role as hosts for pathogenic bacteria and as agents of human disease. In particular, N. fowleri is known to cause the disease primary amoebic meningoencephalitis (PAM) and can be found in drinking water systems in many countries. Understanding the temporal dynamics in relation to environmental and biological factors is vital for developing management tools for mitigating the risks of PAM. Characterizing drinking water systems in Western Australia with a combination of physical, chemical and biological measurements over the course of a year showed a close association of N. fowleri with free chlorine and distance from treatment over the course of a year. This information can be used to help design optimal management strategies for the control of N. fowleri in drinking-water-distribution systems.

Reduced Efficiency of Chlorine Disinfection of Naegleria fowleri in a Drinking Water Distribution Biofilm.

Naegleria fowleri associated with biofilm and biological demand water (organic matter suspended in water that consumes disinfectants) sourced from operational drinking water distribution systems (DWDSs) had significantly increased resistance to chlorine disinfection. N. fowleri survived intermittent chlorine dosing of 0.6 mg/L for 7 days in a mixed biofilm from field and laboratory-cultured Escherichia coli strains. However, N. fowleri associated with an attached drinking water distribution biofilm survived more than 30 times (20 mg/L for 3 h) the recommended concentration of chlorine for drinking water. N. fowleri showed considerably more resistance to chlorine when associated with a real field biofilm compared to the mixed laboratory biofilm. This increased resistance is likely due to not only the consumption of disinfectants by the biofilm and the reduced disinfectant penetration into the biofilm but also the composition and microbial community of the biofilm itself. The increased diversity of the field biofilm community likely increased N. fowleri's resistance to chlorine disinfection compared to that of the laboratory-cultured biofilm. Previous research has been conducted in only laboratory scale models of DWDSs and laboratory-cultured biofilms. To the best of our knowledge, this is the first study demonstrating how N. fowleri can persist in a field drinking water distribution biofilm despite chlorination.

Simultaneous phosphorus uptake and denitrification by EBPR-r biofilm under aerobic conditions: effect of dissolved oxygen.

A biofilm process, termed enhanced biological phosphorus removal and recovery (EBPR-r), was recently developed as a post-denitrification approach to facilitate phosphorus (P) recovery from wastewater. Although simultaneous P uptake and denitrification was achieved despite substantial intrusion of dissolved oxygen (DO >6 mg/L), to what extent DO affects the process was unclear. Hence, in this study a series of batch experiments was conducted to assess the activity of the biofilm under various DO concentrations. The biofilm was first allowed to store acetate (as internal storage) under anaerobic conditions, and was then subjected to various conditions for P uptake (DO: 0-8 mg/L; nitrate: 10 mg-N/L; phosphate: 8 mg-P/L). The results suggest that even at a saturating DO concentration (8 mg/L), the biofilm could take up P and denitrify efficiently (0.70 mmol e(-)/g total solids*h). However, such aerobic denitrification activity was reduced when the biofilm structure was physically disturbed, suggesting that this phenomenon was a consequence of the presence of oxygen gradient across the biofilm. We conclude that when a biofilm system is used, EBPR-r can be effectively operated as a post-denitrification process, even when oxygen intrusion occurs.

Ammonium-oxidizing bacteria facilitate aerobic degradation of sulfanilic acid in activated sludge.

Sulfanilic acid (SA) is a toxic sulfonated aromatic amine commonly found in anaerobically treated azo dye contaminated effluents. Aerobic acclimatization of SA-degrading mixed microbial culture could lead to co-enrichment of ammonium-oxidizing bacteria (AOB) because of the concomitant release of ammonium from SA oxidation. To what extent the co-enriched AOB would affect SA oxidation at various ammonium concentrations was unclear. Here, a series of batch kinetic experiments were conducted to evaluate the effect of AOB on aerobic SA degradation in an acclimatized activated sludge culture capable of oxidizing SA and ammonium simultaneously. To account for the effect of AOB on SA degradation, allylthiourea was used to inhibit AOB activity in the culture. The results indicated that specific SA degradation rate of the mixed culture was negatively correlated with the initial ammonium concentration (0-93 mM, R²= 0.99). The presence of AOB accelerated SA degradation by reducing the inhibitory effect of ammonium (≥ 10 mM). The Haldane substrate inhibition model was used to correlate substrate concentration (SA and ammonium) and oxygen uptake rate. This study revealed, for the first time, that AOB could facilitate SA degradation at high concentration of ammonium (≥ 10 mM) in an enriched activated sludge culture.

Simulating compatible solute biosynthesis using a metabolic flux model of the biomining acidophile, Acidithiobacillus ferrooxidans ATCC 23270.

Halotolerant, acidophilic, bioleaching microorganisms are crucial to biomining operations that utilize saline water. Compatible solutes play an important role in the adaptation of these microorganisms to saline environments. Acidithiobacillus ferrooxidans ATCC 23270, an iron- and sulfur-oxidizing acidophilic bacterium, synthesizes trehalose as its native compatible solute but is still sensitive to salinity. Recently, halotolerant bioleaching bacteria were found to use ectoine as their key compatible solute. Previously, bioleaching bacteria were recalcitrant to genetic manipulation; however, recent advancements in genetic tools and techniques allow successful genetic modification of A. ferrooxidans ATCC 23270. Therefore, this study aimed to test, in silico, the effect of native and synthetic compatible solute biosynthesis by A. ferrooxidans ATCC 23270 on its growth and metabolism. Metabolic network flux modelling was used to provide a computational framework for the prediction of metabolic fluxes during production of native and synthetic compatible solutes by A. ferrooxidans ATCC 23270, in silico. Complete pathways for trehalose biosynthesis by the bacterium are proposed and captured in the updated metabolic model including a newly discovered UDP-dependent trehalose synthesis pathway. Finally, the effect of nitrogen sources on compatible solute production was simulated and showed that using nitrogen gas as the sole nitrogen source enables the ectoine-producing 'engineered' microbe to oxidize up to 20% more ferrous iron in comparison to the native microbe that only produces trehalose. Therefore, the predictive outcomes of the model have the potential to guide the design and optimization of a halotolerant strain of A. ferrooxidans ATCC 23270 for saline bioleaching operations.

Sequential hydrotalcite precipitation, microbial sulfate reduction and in situ hydrogen sulfide removal for neutral mine drainage treatment.

This study proposed and examined a new process flowsheet for treating neutral mine drainage (NMD) from an open-pit gold mine. The process consisted of three sequential stages: (1) in situ hydrotalcite (HT) precipitation; (2) low-cost carbon substrate driven microbial sulfate reduction; and (3) ferrosol reactive barrier for removing biogenic dissolved hydrogen sulfide (H2S). For concept validation, laboratory-scale columns were established and operated for a 140-days period with key process performance parameters regularly measured. At the end, solids recovered from various depths of the ferrosol column were analysed for elemental composition and mineral phases. Prokaryotic microbial communities in various process locations were characterised using 16S rRNA gene sequencing. Results showed that the Stage 1 HT-treatment substantially removed a range of elements (As, B, Ba, Ca, F, Zn, Si, and U) in the NMD, but not nitrate or sulfate. The Stage 2 sulfate reducing bioreactor (SRB) packed with 70 % (v/v) Eucalyptus woodchip, 1 % (w/v) ground (<1 mm) dried Typha biomass, and 10 % (w/v) NMD-pond sediment facilitated complete nitrate removal and stable sulfate removal of ca. 50 % (50 g-SO4 m-3 d-1), with an average H2S generation rate of 10 g-H2S m-3d-1. The H2S-removal performance of the Stage 3 ferrosol column was compared with a synthetic amorphous Fe-oxyhydroxide-amended sand control column. Although both columns facilitated excellent (95-100 %) H2S removal, the control column only enabled a further ca. 10 % sulfate reduction, giving an overall sulfate removal of 56 %. In contrast, the ferrosol enabled an extra 99.9 % sulfate reduction in the SRB effluent, leading to a near complete sulfate removal. Overall, the process successfully eliminated a range of metal/metalloid contaminants, nitrate, sulfate (2500 mg-SO4 L-1 in the NMD to <10 mg-SO4 L-1 in the final effluent) and H2S (>95 % removal). Further optimisation is required to minimise release of ferrous iron from the ferrosol barrier into the final effluent.

Prokaryotic microbial ecology as an ecosurveillance tool for eukaryotic pathogen colonisation: Meiothermus and Naegleria fowleri.

Naegleria fowleri has been detected in drinking water distribution systems (DWDS) in Australia, Pakistan and the United States and is the causative agent of the highly fatal disease primary amoebic meningoencephalitis. Previous small scale field studies have shown that Meiothermus may be a potential biomarker for N. fowleri. However, correlations between predictive biomarkers in small sample sizes often breakdown when applied to larger more representative datasets. This study represents one of the largest and most rigorous temporal investigations of Naegleria fowleri colonisation in an operational DWDS in the world and measured the association of Meiothermus and N. fowleri over a significantly larger space and time in the DWDS. A total of 232 samples were collected from five sites over three-years (2016-2018), which contained 29 positive N. fowleri samples. Two specific operational taxonomic units assigned to M. chliarophilus and M. hypogaeus, were significantly associated with N. fowleri presence. Furthermore, inoculation experiments demonstrated that Meiothermus was required to support N. fowleri growth in field-collected biofilms. This validates Meiothermus as prospective biological tool to aid in the identification and surveillance of N. fowleri colonisable sites.

Biological phosphorus and nitrogen removal in sequencing batch reactors: effects of cycle length, dissolved oxygen concentration and influent particulate matter.

Removal of phosphorus (P) and nitrogen (N) from municipal wastewaters is required to mitigate eutrophication of receiving water bodies. While most treatment plants achieve good N removal using influent carbon (C), the use of influent C to facilitate enhanced biological phosphorus removal (EBPR) is poorly explored. A number of operational parameters can facilitate optimum use of influent C and this study investigated the effects of cycle length, dissolved oxygen (DO) concentration during aerobic period and influent solids on biological P and N removal in sequencing batch reactors (SRBs) using municipal wastewaters. Increasing cycle length from 3 to 6 h increased P removal efficiency, which was attributed to larger portion of N being removed via nitrite pathway and more biodegradable organic C becoming available for EBPR. Further increasing cycle length from 6 to 8 h decreased P removal efficiencies as the demand for biodegradable organic C for denitrification increased as a result of complete nitrification. Decreasing DO concentration in the aerobic period from 2 to 0.8 mg L(-1) increased P removal efficiency but decreased nitrification rates possibly due to oxygen limitation. Further, sedimented wastewater was proved to be a better influent stream than non-sedimented wastewater possibility due to the detrimental effect of particulate matter on biological nutrient removal.

Klebsiella aerogenes Adhesion Behaviour during Biofilm Formation on Monazite.

The adsorption behaviour of micro-organisms during the initial attachment stage of biofilm formation affects subsequent stages. The available area for attachment and the chemophysical properties of a surface affect microbial attachment performance. This study focused on the initial attachment behaviour of Klebsiella aerogenes on monazite by measuring the ratio of planktonic against sessile subpopulations (P:S ratio), and the potential role of extracellular DNA (eDNA). eDNA production, effects of physicochemical properties of the surface, particle size, total available area for attachment, and the initial inoculation size on the attachment behaviour were tested. K. aerogenes attached to monazite immediately after exposure to the ore; however, the P:S ratio significantly (p = 0.05) changed in response to the particle size, available area, and inoculation size. Attachment occurred preferentially on larger-sized (~50 µm) particles, and either decreasing the inoculation size or increasing the available area further promoted attachment. Nevertheless, a portion of the inoculated cells always remained in a planktonic state. K. aerogenes produced lower eDNA in response to the changed surface chemical properties when monazite was replaced by xenotime. Using pure eDNA to cover the monazite surface significantly (p ≤ 0.05) hindered bacterial attachment due to the repulsive interaction between the eDNA layer and bacteria.

Biofilm formation on the surface of monazite and xenotime during bioleaching.

Microbial attachment and biofilm formation is a ubiquitous behaviour of microorganisms and is the most crucial prerequisite of contact bioleaching. Monazite and xenotime are two commercially exploitable minerals containing rare earth elements (REEs). Bioleaching using phosphate solubilizing microorganisms is a green biotechnological approach for the extraction of REEs. In this study, microbial attachment and biofilm formation of Klebsiella aerogenes ATCC 13048 on the surface of these minerals were investigated using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). In a batch culture system, K. aerogenes was able to attach and form biofilms on the surface of three phosphate minerals. The microscopy records showed three distinctive stages of biofilm development for K. aerogenes commencing with initial attachment to the surface occurring in the first minutes of microbial inoculation. This was followed by colonization of the surface and formation of a mature biofilm as the second distinguishable stage, with progression to dispersion as the final stage. The biofilm had a thin-layer structure. The colonization and biofilm formation were localized toward physical surface imperfections such as cracks, pits, grooves and dents. In comparison to monazite and xenotime crystals, a higher proportion of the surface of the high-grade monazite ore was covered by biofilm which could be due to its higher surface roughness. No selective attachment or colonization toward specific mineralogy or chemical composition of the minerals was detected. Finally, in contrast to abiotic leaching of control samples, microbial activity resulted in extensive microbial erosion on the high-grade monazite ore.