Outer Biofilm Research Articles

Simultaneous carbon, nitrogen and phosphorus removal in sequencing batch membrane aerated biofilm reactor with biofilm thickness control via air scouring aided by computational fluid dynamics

Membrane aerated biofilm reactor (MABR) is challenged by biofilm thickness control and phosphorus removal. Air scouring aided by computational fluid dynamics (CFD) was employed to detach outer biofilm in sequencing batch MABR treating low C/N wastewater. Biofilm with 177–285 µm thickness in cycle 5–15 achieved over 85 % chemical oxygen demand (COD) and total inorganic nitrogen (TIN) removals at loading rate of 13.2 gCOD/m2/d and 2.64 gNH4+-N/m2/d. Biofilm rheology measurements in cycle 10–25 showed yield stress against detachment of 2.8–7.4 Pa, which were equal to CFD calculated shear stresses under air scouring flowrate of 3–9 L/min. Air scouring reduced effluent NH4+-N by 10 % and biofilm thickness by 78 µm. Intermittent aeration (4h off, 19.5h on) and air scouring (3 L/min, 30 s before settling) in one cycle achieved COD removal over 90 %, TIN and PO43−-P removals over 80 %, showing great potential for simultaneous carbon, nitrogen and phosphorus removals.

High-resolution 3D spatial distribution of complex microbial colonies revealed by mass spectrometry imaging

IntroductionBacterial living states and the distribution of microbial colony signaling molecules are widely studied using mass spectrometry imaging (MSI). However, current approaches often treat 3D colonies as flat 2D disks, inadvertently omitting valuable details. The challenge of achieving 3D MSI in biofilms persists due to the unique properties of microbial samples. ObjectivesThe study aimed to develop a new biofilm sample preparation method that can realize high-resolution 3D MSI of bacterial colonies to reveal the spatial organization of bacterial colonies. MethodsThis article introduces the moisture-assisted cryo-section (MACS) method, enabling embedding-free sectioning parallel to the growth plane. The MACS method secures intact sections by controlling ambient humidity and slice thickness, preventing molecular delocalization. ResultsCombined with matrix-assisted laser desorption ionization mass spectrometry (MALDI)-MSI, the MACS method provides high-resolution insights into endogenic and exogenous molecule distributions in Pseudomonas aeruginosa (P. aeruginosa) biofilms, including isomeric pairs. Moreover, analyzed colonies are revived into 3D models, vividly depicting molecular distribution from inner to outer layers. Additionally, we investigated metabolite spatiotemporal dynamics in multiple colonies, observing changes over time and distinct patterns in single versus merged colonies. These findings shed light on the repel-merge process for multi-colony formation. Furthermore, our study monitored chemical responses inside biofilms after antibiotic treatment, showing increased antibiotic levels in the outer biofilm layer over time while maintaining low levels in the inner region. Moreover, the MACS method demonstrated its universality and applicability to other bacterial strains. ConclusionThese results unveil complex cell activities within biofilm colonies, offering insights into microbe communities. The MACS method is universally applicable to loosely packed microorganism colonies, overcoming the limitations of previously reported MSI methods. It has great potential for studying bacterial-infected cancer tissues and artificial organs, making it a valuable tool in microbiological research.

Anammox-Mediated Hydroxyapatite Granules: Physicochemical Properties, 3D Hierarchy, and Biofilm Thickness.

Biomineralization inspired the development of simultaneous biological transformations and chemical precipitation for simultaneous nitrogen removal and phosphorus recovery from wastewater, which could compensate for the incapacity of phosphorus management in the new biological route of anaerobic ammonium oxidation (anammox). In this study, we strengthened anammox-mediated biomineralization by long-term feeding of concentrated N, P, and Ca substrates, and a self-assembled matrix of anammox bacteria and hydroxyapatite (HAP) was fabricated in a granular shape, defined as HAP-anammox granules. HAP was identified as the dominant mineral using elemental analysis, X-ray diffraction, and Raman spectroscopy. The intensive precipitation of HAP resulted in a higher inorganic fraction and substantially improved settleability of anammox biomass, which facilitated HAP precipitation by acting as nucleation and metabolically elevated pH. By using X-ray microcomputed tomography, we visually represented the hybrid texture of interwoven HAP pellets and biomass, the core-shell layered architecture of different-sized HAP-anammox granules, and their homogeneously regulated thickness of the outer biofilm (from 118 to 635 μm). This unique architecture endows HAP-anammox granules with outstanding settleability, active biofilm, and tightly bonded biofilm with the carrier, which may explain the excellent performance of these HAP-anammox granules under various challenging operational conditions in previous studies.

Insights into simultaneous nitrogen and phosphorus removal in biofilm: The overlooked comammox Nitrospira and the positive role of glycogen-accumulating organisms

Simultaneous nitrogen and phosphorus removal (SNPR) biofilm system is an effective wastewater treatment process. However, the understanding on the mechanism of functional microorganisms driving SNPR is still limited, especially the role of complete ammonia oxidation (comammox) Nitrospira and glycogen-accumulating organisms (GAO). In this study, a sequencing batch biofilm reactor (SBBR) performing SNPR was operated for 249 d. Based on the 16S rRNA gene, comammox amoA amplicon sequencing, metagenomics and batch experiment, we found that comammox Nitrospira was the main ammonia-oxidizing microorganisms (AOM) and provided nitrite for anaerobic ammonia oxidation (anammox) bacteria (AnAOB). Besides, GAO was dominated by the bacteria of genus Defluviicoccus and played a primary role in reducing nitrate rather than nitrite. Fluorescent in situ hybridization (FISH) analysis confirmed that Nitrospira was enriched in the inner layer of the biofilm. Thus, we put forward a novel insight into the mechanism of SNPR biofilm system. Comammox Nitrospira was responsible for nitrite and nitrate production in the inner biofilm, and AnAOB consumed the produced nitrite during the anammox process. While GAO reduced nitrate to nitrite and polyphosphate-accumulating organisms (PAO) converted nitrite to dinitrogen via denitrifying phosphorus removal in the outer biofilm. These findings provide a new understanding in SNPR biofilm system.

Model-based identification and testing of appropriate strategies to minimize N2O emissions from biofilm deammonification.

Based on a one-year pilot plant operation of a two-step biofilm nitritation-anammox pilot plant, N2O mitigation strategies were identified by applying a newly developed biofilm modeling approach. Due to adapted plant operation, the N2O emission could be diminished by 75% (8.8% → 2.3% of NH4-Noxidized_AOB). The results (measurement and simulation) confirm the huge importance of denitrification as an N2O source or N2O sink, depending on the boundary conditions. A significant reduction of N2O emissions could only be achieved with a one-step deammonification system, which is related to low nitrite and HNO2 concentrations. Increased oxygen concentrations in the bulk phase are not related to decreased emissions. N2O formation by ammonium-oxidizing bacteria (AOB) just shifts deeper into the biofilm; zones with low oxygen concentrations are not avoidable in biofilm systems. Low oxygen concentrations in the bulk phase, however, result in a reduction of the total net N2O formation due to increased activity of heterotrophic bacteria directly at the source of N2O formation (outer biofilm layer). For the model-based identification of mitigation strategies, the standard modeling approaches for biofilms were expanded by including the factor-based N2O formation and emission approach. The new model 'Biofilm/N2OISAH' was successfully validated using data from pilot-scale measurement campaigns. Altogether, the investigation confirms that the employed digital model can strongly support the development of N2O mitigation strategies without the need for specialized measurement inside the biofilm.

Antibiofilm Efficacy of the Pseudomonas aeruginosa Pbunavirus vB_PaeM-SMS29 Loaded onto Dissolving Polyvinyl Alcohol Microneedles

Resistant bacteria prevail in most chronic skin wounds and other biofilm-related topical skin infections. Bacteriophages (phages) have proven their antimicrobial effectiveness for treating different antibiotic-resistant and multidrug-resistant bacterial infections, but not all phages are effective against biofilms. Phages possessing depolymerases can reach different biofilm layers; however, those that do not have depolymerase activity struggle to penetrate and navigate in the intricate 3D biofilm structure and mainly infect bacteria lodged in the outer biofilm layers. To address this, Pseudomonas aeruginosa phage vB_PaeM-SMS29, a phage with poor antibiofilm properties, was incorporated into polyvinyl alcohol (PVA, Mowiol 4:88) supplemented with 0.1% (v/v) of glycerol, and cast onto two different microneedle arrays varying in geometry. The dissolving microneedles were thoroughly characterized by microscopy, force-displacement, swelling, phage release and stability. Furthermore, 48 h-old biofilms were formed using the colony biofilm procedure (absence of broth), and the antibiofilm efficacy of the phage-loaded microneedles was evaluated by viable cell counts and microscopy and compared to free phages. The phages in microneedles were fairly stable for six months when stored at 4 °C, with minor decreases in phage titers observed. The geometry of the microneedles influenced the penetration and force-displacement characteristics but not the antimicrobial efficacy against biofilms. The two PVA microneedles loaded with phages reduced P. aeruginosa PAO1 biofilms by 2.44 to 2.76 log10 CFU·cm−2 at 24 h. These values are significantly higher than the result obtained after the treatment with the free phage (1.09 log10 CFU·cm−2). Overall, this study shows that the distribution of phages caused by the mechanical disruption of biofilms using dissolving microneedles can be an effective delivery method against topical biofilm-related skin infections.

Oral biofilm uptake of mineral ions released from experimental toothpaste containing surface pre-reacted glass-ionomer (S-PRG) filler

To clarify the fluoride/mineral kinetics in an oral biofilm following concurrent application of fluoride and other mineral ions released from experimental toothpaste containing S-PRG filler using depth-specific analysis. Twenty subjects wore in situ plaque-generating devices, comprised of a pair of enamel slabs, and a biofilm was allowed to form. The devices were removed after three days, immersed in the toothpaste filtrate containing Al, B, Sr and F ions for 1 min, and then reinserted at the same location. After 30 min, the devices were removed and samples were obtained by sectioning into outer, middle and inner biofilm layers (300-μm thick). Samples treated with filtrate containing F without S-PRG filler extract served as the control. Fluoride and the three other mineral ions extracted from 4-μm sections were quantified using a fluoride electrode and ICP-AES, respectively. The results were corrected for biomass volume, estimated by the area measurement of stained 2-μm sections. The mean uptake ratios (S-PRG/control, ng/mm3) of Al, B, Sr and F were 186.6/53.7, 58.4/25.0, 456.9/125.7 and 43.6/12.0, respectively, in the outer layer, indicating that the mineral ions could easily diffuse into the biofilm. F concentrations in the outer biofilm treated using filtrate with S-PRG filler extract were significantly higher than those in controls, although both biofilms were exposed to filtrates containing the same level of F. The results suggest that toothpaste containing S-PRG filler promotes fluoride retention in oral biofilms via the uptake of other mineral ions.

Cow manure anaerobic fermentation effluent treatment by oxygen-based membrane aerated biofilm reactor

After cow manure undergoes anaerobic fermentation process, it still contains several pollutants such as nitrogen, phosphorus, and organic matter. If not treated properly, these pollutants would cause secondary pollution. Membrane-aerated biofilm reactor (MABR) has been widely used to handle the wastewater with high concentration contaminants. In this study, MABR was utilized to treat the cow manure anaerobic fermentation effluence (CMAFE). In order to avoid incomplete biofilm being affected by high concentration of CMAFE, a gradient dilution of CMAFE was employed. The removal rates of NH4+-N and COD were achieved up to 90% and 85%, respectively, in different dilution ratios. RDA demonstrated that NH4+-N removal was in a full-nitrification process in higher dilution ratio (Stage 1 and 2) but in short-nitrification in lower dilution ratio (Stage 4). The gradient dilution did not affect the biofilm growth. The Inner biofilm contained higher amounts of extracellular polymeric substance (EPS) than the Outer biofilm, which reduced the impact of high-concentration CMAFE on the internal aerobic bacteria, especially nitrifying bacteria. Concerning bacteria abundance, a significant modification occurred in the biofilm compared to raw sludge, demonstrating that MABR had a great potential in CMAFE treatment.

The Effect of Bioinduced Increased pH on the Enrichment of Calcium Phosphate in Granules during Anaerobic Treatment of Black Water.

Simultaneous recovery of calcium phosphate granules (CaP granules) and methane in anaerobic treatment of source separated black water (BW) has been previously demonstrated. The exact mechanism behind the accumulation of calcium phosphate (Cax(PO4)y) in CaP granules during black water treatment was investigated in this study by examination of the interface between the outer anaerobic biofilm and the core of CaP granules. A key factor in this process is the pH profile in CaP granules, which increases from the edge (7.4) to the center (7.9). The pH increase enhances supersaturation for Cax(PO4)y phases, creating internal conditions preferable for Cax(PO4)y precipitation. The pH profile can be explained by measured bioconversion of acetate and H2, HCO3– and H+ into CH4 in the outer biofilm and eventual stripping of CO2 and CH4 (biogas) from the granule. Phosphorus content and Cax(PO4)y crystal mass quantity in the granules positively correlated with the granule size, in the reactor without Ca2+ addition, indicating that the phosphorus rich core matures with the granule growth. Adding Ca2+ increased the overall phosphorus content in granules >0.4 mm diameter, but not in fine particles (<0.4 mm). Additionally, H+ released from aqueous phosphate species during Cax(PO4)y crystallization were buffered by internal hydrogenotrophic methanogenesis and stripping of biogas from the granule. These insights into the formation and growth of CaP granules are important for process optimization, enabling simultaneous Cax(PO4)y and CH4 recovery in a single reactor. Moreover, the biological induction of Cax(PO4)y crystallization resulting from biological increase of pH is relevant for stimulation and control of (bio)crystallization and (bio)mineralization in real environmental conditions.

Sonobactericide - An Adjunct Therapy for Infective Endocarditis: An In Vitro Proof-of-Concept

Infective endocarditis (IE) is associated with high morbidity and mortality rates. The predominant bacteria causing IE is Staphylococcus aureus (S. aureus), which can bind to existing thrombi on heart valves and generate vegetations (biofilms). In this in vitro study, we evaluated sonobactericide as a novel strategy to treat IE, using ultrasound and an ultrasound contrast agent in combination with an antibiotic and a thrombolytic. We developed a model of IE vegetations using human whole-blood clots infected with S. aureus. Histology and live-cell imaging revealed an outer biofilm layer of fibrin-embedded living Staphylococci around a dense erythrocyte core. Infected clots were treated under flow for 30 minutes and degradation was assessed by time-lapse microscopy imaging. Treatments consisted of either continuous plasma flow alone or with different combinations of therapeutics: oxacillin (antibiotic), recombinant tissue plasminogen activator (rt-PA; thrombolytic), intermittent continuous-wave low-frequency ultrasound (120-kHz, 0.44 MPa peak-to-peak pressure), and an ultrasound contrast agent (Definity®). Infected clots exposed to the combination of oxacillin, rt-PA, ultrasound, and Definity achieved 99.3 ± 1.7% clot width loss, which was greater than the other treatment arms. Size measurements of the effluent suggested low emboli risk. These results demonstrate that sonobactericide could be an effective therapeutic strategy for treating IE.

Short and long term biosorption of silica-coated iron oxide nanoparticles in heterotrophic biofilms

The increased application of engineered nanoparticles (ENP) in industrial processes and consumer products has raised concerns about their impact on health and environmental safety. When ENP enter the global water cycle by e.g. wastewater streams, wastewater treatment plants (WWTP) represent potential sinks for ENP. During biological WWT, the attachment of ENP to biofilms is responsible for the desired removal of ENP from the water phase avoiding their release into the aquatic environment. However, the fundamental mechanisms guiding the interactions between ENP and biofilms are not yet fully understood. Therefore, this study investigates the behavior and biosorption of inorganic ENP, here magnetic iron oxide nanoparticles coated with silica (scFe3O4-NP), with heterotrophic biofilms at different time scales. Their magnetic properties enable to follow scFe3O4-NP in the biofilm system by a magnetic susceptibility balance and magnetic resonance imaging. Biofilms were exposed to scFe3O4-NP at short contact times (5min) in flow cells and complementary, scFe3O4-NP were introduced into a moving bed biofilm reactor (MBBR) to be observed for 27 d. Mass balances revealed that scFe3O4-NP sorbed to the biofilm within a few minutes, but that the total biosorption was rather low (3.2μgFe/mg TSS). scFe3O4-NP mainly sorbed to the biofilm surface inducing the detachment of outer biofilm parts starting after an exposure time of 3h in the MBBR. The biosorption depended on the exposure concentration of scFe3O4-NP, but less on the contact time. Most scFe3O4-NP exited the flow cell (up to 65%) and the MBBR (57%) via the effluent. This effect was favored by the stabilization of scFe3O4-NP in the bulk liquid by organic matter leading to a low retention capacity of the MBBR system. The results contribute to improve our understanding about the fate of ENP in environmental and in technical biofilm systems and give indications for future investigations needed.

Nitrous oxide emissions in a biofilm loaded with different mixtures of concentrated household wastewater

Aerobic biofilm systems are increasingly used for household wastewater treatment, but little is known about their potential to emit nitrous oxide in response to different loading conditions. We studied nitrification and nitrous oxide production in a biofilm reactor that was continuously fed with three different mixtures of household wastewater. Higher proportions of blackwater increased nitrification activity, which resulted in enhanced nitrite accumulation and nitrous oxide emissions. Applying a conceptual biofilm model together with the results of ancillary batch incubations suggested that this was caused by a higher proportion of slowly degradable compounds in blackwater. Increasing amounts of blackwater would result in less oxygen depletion by heterotrophic degradation in outer biofilm layers, leading to nitrite accumulation by enhanced ammonia oxidation as well as electron limitation of denitrification in anoxic biofilm layers. Under such conditions, nitrifier denitrification and incomplete heterotrophic denitrification would be the prevailing sources of nitrous oxide emission. These assumptions are supported by an exponential increase in the determined emission factor (nitrous oxide relative to oxidized ammonia), which accounted for 0.7 with 20 % blackwater, 1.1 with 50 % blackwater, and 8.5 with 100 % blackwater in the wastewater load.

Abundance of the multiheme c-type cytochrome OmcB increases in outer biofilm layers of electrode-grown Geobacter sulfurreducens.

When Geobacter sulfurreducens utilizes an electrode as its electron acceptor, cells embed themselves in a conductive biofilm tens of microns thick. While environmental conditions such as pH or redox potential have been shown to change close to the electrode, less is known about the response of G. sulfurreducens to growth in this biofilm environment. To investigate whether respiratory protein abundance varies with distance from the electrode, antibodies against an outer membrane multiheme cytochrome (OmcB) and cytoplasmic acetate kinase (AckA) were used to determine protein localization in slices spanning ∼25 µm-thick G. sulfurreducens biofilms growing on polished electrodes poised at +0.24 V (vs. Standard Hydrogen Electrode). Slices were immunogold labeled post-fixing, imaged via transmission electron microscopy, and digitally reassembled to create continuous images allowing subcellular location and abundance per cell to be quantified across an entire biofilm. OmcB was predominantly localized on cell membranes, and 3.6-fold more OmcB was detected on cells 10–20 µm distant from the electrode surface compared to inner layers (0–10 µm). In contrast, acetate kinase remained constant throughout the biofilm, and was always associated with the cell interior. This method for detecting proteins in intact conductive biofilms supports a model where the utilization of redox proteins changes with depth.

Microanatomy at Cellular Resolution and Spatial Order of Physiological Differentiation in a Bacterial Biofilm

ABSTRACTBacterial biofilms are highly structured multicellular communities whose formation involves flagella and an extracellular matrix of adhesins, amyloid fibers, and exopolysaccharides. Flagella are produced by still-dividing rod-shaped Escherichia coli cells during postexponential growth when nutrients become suboptimal. Upon entry into stationary phase, however, cells stop producing flagella, become ovoid, and generate amyloid curli fibers. These morphological changes, as well as accompanying global changes in gene expression and cellular physiology, depend on the induction of the stationary-phase sigma subunit of RNA polymerase, σS (RpoS), the nucleotide second messengers cyclic AMP (cAMP), ppGpp, and cyclic-di-GMP, and a biofilm-controlling transcription factor, CsgD. Using flagella, curli fibers, a CsgD::GFP reporter, and cell morphology as “anatomical” hallmarks in fluorescence and scanning electron microscopy, different physiological zones in macrocolony biofilms of E. coli K-12 can be distinguished at cellular resolution. Small ovoid cells encased in a network of curli fibers form the outer biofilm layer. Inner regions are characterized by heterogeneous CsgD::GFP and curli expression. The bottom zone of the macrocolonies features elongated dividing cells and a tight mesh of entangled flagella, the formation of which requires flagellar motor function. Also, the cells in the outer-rim growth zone produce flagella, which wrap around and tether cells together. Adjacent to this growth zone, small chains and patches of shorter curli-surrounded cells appear side by side with flagellated curli-free cells before curli coverage finally becomes confluent, with essentially all cells in the surface layer being encased in “curli baskets.”

Quantitative evaluation of the oral biofilm-removing capacity of a dental water jet using an electron-probe microanalyzer

ObjectiveThis study was conducted to evaluate the oral biofilm-removing capacity of a dental water jet (DWJ) by measuring biofilm thickness using an electron-probe microanalyzer (EPMA). MethodsThirty consenting subjects wore in situ plaque-generating devices, which consisted of a pair of 4mm2 enamel slabs attached to the upper molars for 2 days. Each device removed from the mouth was clamped, and one of the slab surfaces was treated with the DWJ, irrigating it for 5s. The devices were randomly assigned to three different pressure settings of 707, 350 or 102kPa. Another slab with no treatment served as a control. Each slab was freeze-dried, sputter-coated with platinum, and examined using secondary-electron imaging. The slabs were then embedded in methacrylate and cross-sectioned in the centre. Their surfaces were polished, coated with carbon, and examined using backscattered electron compositional (COMPO) imaging. The area between the enamel and the outer biofilm surface, indicated by a thin platinum layer, was measured by COMPO imaging to calculate the average thickness of the biofilm on the specimen. ResultsThe removal capacity of biofilm by irrigation was estimated using a reduced rate of biofilm thickness, which was calculated from the differences between a pair of treated and control slabs. The reduced rates were 85.5% at 707kPa, 85.1% at 350kPa and 63.4% at 102kPa, indicating that biofilm thickness was significantly reduced at every pressure setting. ConclusionsThe results suggest that irrigation using a DWJ would be an effective means of plaque control.

Microtoming coupled to microarray analysis to evaluate the spatial metabolic status of Geobacter sulfurreducens biofilms

Further insight into the metabolic status of cells within anode biofilms is essential for understanding the functioning of microbial fuel cells and developing strategies to optimize their power output. Cells throughout anode biofilms of Geobacter sulfurreducens reduced the metabolic stains: 5-cyano-2,3-ditolyl tetrazolium chloride and Redox Green, suggesting metabolic activity throughout the biofilm. To compare the metabolic status of cells growing close to the anode versus cells in the outer portion of the anode biofilm, anode biofilms were encased in resin and sectioned into inner (0-20 microm from anode surface) and outer (30-60 microm) fractions. Transcriptional analysis revealed that, at a twofold threshold, 146 genes had significant (P<0.05) differences in transcript abundance between the inner and outer biofilm sections. Only 1 gene, GSU0093, a hypothetical ATP-binding cassette transporter, had significantly higher transcript abundances in the outer biofilm. Genes with lower transcript abundance in the outer biofilm included genes for ribosomal proteins and NADH dehydrogenase, suggesting lower metabolic rates. However, differences in transcript abundance were relatively low (<threefold) and the expression of genes for the tricarboxylic acid cycle enzymes was not significantly lower. Lower expression of genes involved in stress responses in the outer biofilm may reflect the development of low pH near the surface of the anode. The results of this study suggest that cells throughout the biofilm are metabolically active and can potentially contribute to current production. The microtoming/microarray strategy described here may be useful for evaluating gene expression with depth in a diversity of microbial biofilms.

Investigation of biofilm growth and attrition in a three‐phase airlift bioreactor using 35SO42− as a radiolabelled tracer

AbstractThe growth and biomass loss pattern for immobilized cells growing on diatomaceous earth particles in a three‐phase airlift bioreactor (TPALB) is studied using 35S as a radiolabelled tracer. A monoculture of Beneckea natriegens was grown immobilized on support particles in a 3 L TPALB. Sterile conditions were maintained during the experiments, and n‐propanol was used as the sole carbon source. After the system reached steady state, the unlabelled sulfate (SO42−) in the feed tank was substituted by a radioactive grade (35SO42−), and the assimilation of 35S in the immobilized and in the suspended cells was monitored. The results indicated that the growth rate of immobilized biomass was not uniform throughout the biofilm. More specifically, it was concluded that cells growing closer to the external biofilm layer exhibit a higher growth rate, and that biomass loss from the biofilm was through attrition at the outer biofilm surface rather than by sloughing off of whole sections of biofilm. Copyright © 2006 Society of Chemical Industry

Integrated anaerobic–aerobic fixed-film reactor for slaughterhouse wastewater treatment

An integrated anaerobic-aerobic fixed-film pilot-scale reactor with arranged media was fed during 166 days with slaughterhouse wastewater. Operation temperature was 25 degrees C and the anaerobic-aerobic volume ratio was decreased from 4:1 to 3:2 and finally to 2:3. Overall organic matter removal efficiencies of 93% were achieved for an average organic loading rate of 0.77 kg COD/m3 d, and nitrogen removal efficiencies of 67% were achieved for nitrogen loading rates of 0.084 kg N/m3 d. The high internal recirculation associated to the air-lift effect linked to the aeration of a part of the reactor section caused high mixing between the anaerobic and aerobic zones, so that most organic matter was removed aerobically. The nitrification process achieved an efficiency of 91% for nitrogen loads of 0.15 kg N/m3 d when the anaerobic-aerobic volume ratio was 2:3 and was limited by dissolved oxygen concentration below 3 mg/l. The influence of the heterotrophic biomass growing in the outer biofilm was checked. Denitrification only implied the 12-34% of the total nitrogen removal and was limited by dissolved oxygen concentration in the anaerobic zone above 0.5 mg/l caused by the mixing regime. Most removed nitrogen was employed in synthesis of heterotrophic bacteria.

Identification and quantification of nitrogen removal in a rotating biological contactor by 15N tracer techniques

High autotrophic nitrogen removal rates of 858 mg N L −1 day −1 or 1.55 g N m −2 day −1 were obtained in a lab-scale rotating biological contactor treating an ammonium rich influent. It was postulated that ammonium was removed as dinitrogen gas by a sequence of aerobic ammonium oxidation to nitrite taking place in the outer biofilm layer and anaerobic ammonium oxidation with nitrite as electron acceptor occuring in the deeper biofilm layer. Chemical evidence for anaerobic ammonium oxidation within intact biofilm sludge from a lab-scale rotating biological contactor could be provided, without direct identification of responsible organisms catalysing this reaction. 15N tracer techniques were used for identification and quantification of nitrogen transformations. In batch tests with biofilm sludge at dissolved oxygen concentrations lower than 0.1 mg L −1, ammonium and nitrite did react in a stoichiometric ratio of 1:1.43 thereby forming dinitrogen. 15N isotope dilution calculations revealed that anaerobic ammonium oxidation was the major nitrogen transformation leading to concomitant ammonium and nitrite removal. Isotopic analysis of the produced biogas showed that both ammonium-N and nitrite-N were incorporated in N 2.

Strategies for augmenting the pentachlorophenol degradation potential of UASB anaerobic granules

Anaerobic degradation of pentachlorophenol (PCP) is an example of a process that may benefit from enrichment or bioaugmentation. In one approach, enrichment acceleration was attempted by applying an on-line control-based selective stress strategy to a native anaerobic upflow sludge bed (UASB) system; this strategy linked PCP loading rate to methane production. As a result, the reactor biomass potential for PCP complete dechlorination reached a rate of 4 mg g(-1) volatile suspended solid (VSS) day(-1) within a period of 120 days. In another approach, a pure culture, Desulfitobacterium frappieri PCP-1, a strictly anaerobic Gram-positive bacterium, was used to augment the granular biomass of the UASB reactor. This also resulted in a specific degradation rate of 4 mg PCPg(-1) VSS day(-1); however, this potential was attained within 56 days. Fluorescent in situ hybridization (FISH) showed that the PCP-1 strain was able to rapidly attach to the granule and densely colonize the outer biofilm layer.