Small-bioreactor platform technology as a municipal wastewater additive treatment.

The bioaugmentation treatment approach presents both an economical and environmentally friendly solution for wastewater treatment. However, the use of exogenous bacterial cultures presents several limitations: negative interaction between microorganisms and adaptation to new physical and chemical composite environment. These selective forces create a significant challenge for the introduced culture to achieving the required biomass in order to conduct the target biological treatment. Small-bioreactor platform (SBP) technology is aimed at introducing exogenous bacterial culture with some protection to reduce some of the natural selection process. The current study was aimed at validating the use of SBP technology to improve biological treatment, especially during a stress period, by using macro-encapsulated bioaugmentation treatment. The study results indicate that the use of SBP technology elevates the stability of biological treatment, improving operational factors such as the reduction of foaming phenomena and sludge accumulation. Still, a significant study needs to be conducted to understand the potential of this technology; especially the impact on biological treatment by using different types of microorganisms for different types of wastewaters and the relationship between the biomass within the SBP capsules and the natural microorganisms.

Selective enrichment, identification, and isolation of diclofenac, ibuprofen, and carbamazepine degrading bacteria from a groundwater biofilm.

Diclofenac, ibuprofen, and carbamazepine are three of the most widely detected and most concerning pharmaceutical residues in aquatic ecosystems. The aim of this study was to identify bacteria that may be involved in their degradation from a bacterial biofilm. Selective enrichment cultures in mineral salt solution containing pharmaceutical compounds as sole source of carbon and energy were set up, and population dynamics were monitored using shotgun metagenome sequencing. Bacterial genomes were reconstructed using genome-resolved metagenomics. Thirty bacterial isolates were obtained, identified at species level, and tested regarding pharmaceutical biodegradation at an initial concentration of 1.5 mg l-1. The results indicated that most probably diclofenac biodegrading cultures consisted of members of genera Ferrovibrio, Hydrocarboniphaga, Zavarzinia, and Sphingopyxis, while in ibuprofen biodegradation Nocardioides and Starkeya, and in carbamazepine biodegradation Nocardioides, Pseudonocardia, and Sphingopyxis might be involved. During the enrichments, compared to the initial state the percentage relative abundance of these genera increased up to three orders of magnitude. Except Starkeya, the genomes of these bacteria were reconstructed and annotated. Metabolic analyses of the annotated genomes indicated that these bacteria harbored genes associated with pharmaceutical biodegradation. Stenotrophomonas humi DIC_5 and Rhizobium daejeonense IBU_18 isolates eliminated diclofenac and ibuprofen during the tests in the presence of either glucose (3 g l-1) or in R2A broth. Higher than 90% concentration reduction was observed in the case of both compounds.

A tailored permeable reactive bio-barrier for in situ groundwater remediation: removal of 3-chlorophenol as a case study.

The present study explored bacterial aerobic biodegradation of reduced carbon-contaminants (RCC) in a pilot system mimicking remediation of a saturated aquifer in a permeable reactive biobarrier (PRBB). Bioaugmentation was performed with a pure culture of Pseudomonas putida macro-encapsulated in a cellulose-acetate porous envelope and integrated transversely to the flow trajectory of the fluid in the biobarrier and compared with controls without capsules. The macro-encapsulation technique applied allowed the incorporation of a built-in nutrient core for the slow release of macronutrients, i.e. N, P, instead of exogenous nutrients supply. 3-Chlorophenol (3CP) at a concentration range of 350-500 mg/L was chosen as an RCC model compound. The findings indicate efficient 3CP biodegradation during the PRBB operation with a similar degree of transformation (76 ± 2% and 72 ± 2%) and mineralization (55 ± 4% vs. 49 ± 3%) for exogenous and built-in nutrients supply, respectively. The extent of dechlorination in both cases (54 ± 10% vs. 40 ± 2%, respectively) followed mineralization rather than transformation, suggesting that Cl- release took place in late transformation stages. Negligible decontamination was observed in the control system without bioaugmentation. Concluding, tailored PRBB with macro-capsules incorporating a built-in nutrient core to support bacterial growth presents a significant environmental advantage controlling excess nutrients release required in bioremediation of oligotrophic systems.

Hydrogen Production in Microbial Electrolysis Cells Based on Bacterial Anodes Encapsulated in a Small Bioreactor Platform.

Microbial electrolysis cells (MECs) are an emerging technology capable of harvesting part of the potential chemical energy in organic compounds while producing hydrogen. One of the main obstacles in MECs is the bacterial anode, which usually contains mixed cultures. Non-exoelectrogens can act as a physical barrier by settling on the anode surface and displacing the exoelectrogenic microorganisms. Those non-exoelectrogens can also compete with the exoelectrogenic microorganisms for nutrients and reduce hydrogen production. In addition, the bacterial anode needs to withstand the shear and friction forces existing in domestic wastewater plants. In this study, a bacterial anode was encapsulated by a microfiltration membrane. The novel encapsulation technology is based on a small bioreactor platform (SBP) recently developed for achieving successful bioaugmentation in wastewater treatment plants. The 3D capsule (2.5 cm in length, 0.8 cm in diameter) physically separates the exoelectrogenic biofilm on the carbon cloth anode material from the natural microorganisms in the wastewater, while enabling the diffusion of nutrients through the capsule membrane. MECs based on the SBP anode (MEC-SBPs) and the MECs based on a nonencapsulated anode (MEC control) were fed with Geobacter medium supplied with acetate for 32 days, and then with artificial wastewater for another 46 days. The electrochemical activity, chemical oxygen demand (COD), bacterial anode viability and relative distribution on the MEC-SBP anode were compared with the MEC control. When the MECs were fed with artificial wastewater, the MEC-SBP produced (at -0.6 V) 1.70 ± 0.22 A m-2, twice that of the MEC control. The hydrogen evolution rates were 0.017 and 0.005 m3 m-3 day-1, respectively. The COD consumption rate for both was about the same at 650 ± 70 mg L-1. We assume that developing the encapsulated bacterial anode using the SBP technology will help overcome the problem of contamination by non-exoelectrogenic bacteria, as well as the shear and friction forces in wastewater plants.

Nocardioides carbamazepini sp. nov., an ibuprofen degrader isolated from a biofilm bacterial community enriched on carbamazepine.

From the metagenome of a carbamazepine amended selective enrichment culture the genome of a new to science bacterial species affiliating with the genus Nocardioides was reconstructed. From the same enrichment an aerobic actinobacterium, strain CBZ_1T, sharing 99.4% whole-genome sequence similarity with the reconstructed Nocardioides sp. bin genome was isolated. On the basis of 16S rRNA gene sequence similarity the novel isolate affiliated to the genus Nocardioides, with the closest relatives Nocardioides kongjuensis DSM19082T (98.4%), Nocardioides daeguensis JCM17460T (98.4%) and Nocardioides nitrophenolicus DSM15529T (98.2%). Using a polyphasic approach it was confirmed that the isolate CBZ_1T represents a new phyletic lineage within the genus Nocardioides. According to metagenomic, metatranscriptomic studies and metabolic analyses strain CZB_1T was abundant in both carbamazepine and ibuprofen enrichments, and harbors biodegradative genes involved in the biodegradation of pharmaceutical compounds. Biodegradation studies supported that the new species was capable of ibuprofen biodegradation. After 7 weeks of incubation, in mineral salts solution supplemented with glucose (3 g l-1) as co-substrate, 70% of ibuprofen was eliminated by strain CBZ_1T at an initial conc. of 1.5 mg l-1. The phylogenetic, phenotypic and chemotaxonomic data supported the classification of strain CBZ_1T to the genus Nocardioides, for which the name Nocardioides carbamazepini sp. nov. (CBZ_1T = NCAIM B.0.2663 = LMG 32395) is proposed. To the best of our knowledge, this is the first study that reports simultaneous genome reconstruction of a new to science bacterial species using metagenome binning and at the same time the isolation of the same novel bacterial species.

UV-LED Combined with Small Bioreactor Platform (SBP) for Degradation of 17α-Ethynylestradiol (EE2) at Very Short Hydraulic Retention Time.

Degradation of 17α-ethynylestradiol (EE2) and estrogenicity were examined in a novel oxidative bioreactor (OBR) that combines small bioreactor platform (SBP) capsules and UV-LED (ultraviolet light emission diode) simultaneously, using enriched water and secondary effluent. Preliminary experiments examined three UV-LED wavelengths-267, 279, and 286 nm, with (indirect photolysis) and without (direct photolysis) H2O2. The major degradation wavelength for both direct and indirect photolysis was 279 nm, while the major removal gap for direct vs. indirect degradation was at 267 nm. Reduction of EE2 was observed together with reduction of estrogenicity and mineralization, indicating that the EE2 degradation products are not estrogens. Furthermore, slight mineralization occurred with direct photolysis and more significant mineralization with the indirect process. The physical-biological OBR process showed major improvement over other processes studied here, at a very short hydraulic retention time. The OBR can feasibly replace the advanced oxidation process of UV-LED radiation with catalyst in secondary sedimentation tanks with respect to reduction ratio, and with no residual H2O2. Further research into this OBR system is warranted, not only for EE2 degradation, but also to determine its capabilities for degrading mixtures of pharmaceuticals and pesticides, both of which have a significant impact on the environment and public health.

Potential of Variovorax paradoxus isolate BFB1_13 for bioremediation of BTEX contaminated sites.

Here, we report and discuss the applicability of Variovorax paradoxus strain BFB1_13 in the bioremediation of BTEX contaminated sites. Strain BFB1_13 was capable of degrading all the six BTEX-compounds under both aerobic (O2 conc. 8 mg l-1) and micro-aerobic/oxygen-limited (O2 conc. 0.5 mg l-1) conditions using either individual (8 mg‧l-1) or a mixture of compounds (~ 1.3 mg‧l-1 of each BTEX compound). The BTEX biodegradation capability of SBP-encapsulated cultures (SBP-Small Bioreactor Platform) was also assessed. The fastest degradation rate was observed in the case of aerobic benzene biodegradation (8 mg l-1 per 90 h). Complete biodegradation of other BTEX occurred after at least 168 h of incubation, irrespective of the oxygenation and encapsulation. No statistically significant difference was observed between aerobic and microaerobic BTEX biodegradation. Genes involved in BTEX biodegradation were annotated and degradation pathways were predicted based on whole-genome shotgun sequencing and metabolic analysis. We conclude that V. paradoxus strain BFB1_13 could be used for the development of reactive biobarriers for the containment and in situ decontamination of BTEX contaminated groundwater plumes. Our results suggest that V. paradoxus strain BFB1_13-alone or in co-culture with other BTEX degrading bacterial isolates-can be a new and efficient commercial bioremediation agent for BTEX contaminated sites.

Salicylate reduces the antimicrobial activity of ciprofloxacin against extracellular Salmonella enterica serovar Typhimurium, but not against Salmonella in macrophages.

OBJECTIVES: Salicylate, a potent inducer of the MarA activator in Salmonella enterica, is the principal metabolite of aspirin, which is often consumed for medicinal and cosmetic uses. Our research was aimed at testing if salicylate activates the mar regulon in macrophage-associated Salmonella (intracellular bacteria), and investigating its effects on bacterial susceptibility to ciprofloxacin extracellularly and intracellularly. METHODS: J774 macrophages were infected with S. enterica serovar Typhimurium (wild-type and marA null mutant), treated with ciprofloxacin with and without pre-exposure to salicylate, and the surviving bacteria were counted. Similar experiments were conducted with bacteria in broth (extracellular bacteria). Phe-Arg-beta-naphthylamide (PAbetaN) was added to investigate the role of efflux pumps in resistance. The transcriptional regulation of marRAB, acrAB and micF in extracellular and intracellular Salmonella Typhimurium with and without salicylate and ciprofloxacin was investigated using green fluorescent protein as a marker protein and quantitative real time PCR. RESULTS: Pre-exposure of Salmonella to salicylate increased the resistance of extracellular but not intracellular bacteria to ciprofloxacin, although salicylate stimulated the expression of mar genes in intracellular and extracellular bacteria. Using marA mutants and the inhibitor PAbetaN, we showed that the improved resistance in extracellular bacteria is derived from the induction of acrAB by salicylate, which is mediated by MarA. CONCLUSIONS: In intracellular bacteria, the expression of acrAB is already higher when compared with extracellular cells; therefore, salicylate does not result in significant acrAB induction intracellularly and subsequent resistance enhancement. Results show that conclusions raised from extracellular studies cannot be applied to intracellular bacteria, although the systems have similar functions.

Phenol biodegradation by bacterial cultures encapsulated in 3D microfiltration-membrane capsules.

The aim of the study was to evaluate the performance of batch and semi-continuous treatment systems for phenol degradation using a consortium of bacterial cultures that were encapsulated using the 'Small Bioreactor Platform' (SBP) encapsulation method. The maximal phenol biodegradation rate was 22 and 48 mg/L/h at an initial phenol concentration of 100 and 1000 mg/L in the batch and semi-continuous bioreactors, respectively. The initial phenol concentration played an important role in the degradation efficiency rates. The batch bioreactor results could be described by the Haldane model, where the degradation rate decreased under low as well as under very high initial phenol concentrations. Concentration equalization between the two sides of the SBP capsule's membrane occurred after 80 min. The microfiltration membrane is perforated with holes that have an average diameter of 0.2-0.7 µm. It is therefore suggested that the capsule's membrane is more permeable compared to other polymeric matrixes used for bacterial encapsulation (such as alginate). This study shows that the encapsulation of phenol degraders within microfiltration-membrane capsules which create a confined environment has a potential for enhancing phenol removal in phenol-rich wastewaters.

LP-UV-Nano MgO2 Pretreated Catalysis Followed by Small Bioreactor Platform Capsules Treatment for Superior Kinetic Degradation Performance of 17α-Ethynylestradiol.

A successful attempt to degrade synthetic estrogen 17α-ethynylestradiol (EE2) is demonstrated via combining photocatalysis employing magnesium peroxide (MgO2)/low-pressure ultraviolet (LP-UV) treatment followed by biological treatment using small bioreactor platform (SBP) capsules. Reusable MgO2 was synthesized through wet chemical synthesis and extensively characterized by X-ray diffraction (XRD) for phase confirmation, X-ray photoelectron spectroscopy (XPS) for elemental composition, Brunauer-Emmett-Teller (BET) to explain a specific surface area, scanning electron microscopy (SEM) imaging surface morphology, and UV-visible (Vis) spectrophotometry. The degradation mechanism of EE2 by MgO2/LP-UV consisted of LP-UV photolysis of H2O2 in situ (produced by the catalyst under ambient conditions) to generate hydroxyl radicals, and the degradation extent depended on both MgO2 and UV dose. Moreover, the catalyst was successfully reusable for the removal of EE2. Photocatalytic treatment by MgO2 alone required 60 min (~1700 mJ/cm2) to remove 99% of the EE2, whereas biodegradation by SBP capsules alone required 24 h to remove 86% of the EE2, and complete removal was not reached. The sequential treatment of photocatalysis and SBP biodegradation to achieve complete removal required only 25 min of UV (~700 mJ/cm2) and 4 h of biodegradation (instead of >24 h). The combination of UV photocatalysis and biodegradation produced a greater level of EE2 degradation at a lower LP-UV dose and at less biodegradation time than either treatment used separately, proving that synergetic photocatalysis and biodegradation are effective treatments for degrading EE2.

Encapsulated Pseudomonas putida for phenol biodegradation: Use of a structural membrane for construction of a well-organized confined particle.

Phenols are toxic byproducts from a wide range of industry sectors. If not treated, they form effluents that are very hazardous to the environment. This study presents the use of a Pseudomonas putida F1 culture encapsulated within a confined environment particle as an efficient technique for phenol biodegradation. The innovative encapsulation technique method, named the "Small Bioreactor Platform" (SBP) technology, enables the use of a microfiltration membrane constructed as a physical barrier for creating a confined environment for the encapsulated culture. The phenol biodegradation rate of the encapsulated culture was compared to its suspended state in order to evaluate the effectiveness of the encapsulation technique for phenol biodegradation. A maximal phenol biodegradation rate (q) of 2.12/d was exhibited by encapsulated P. putida at an initial phenol concentration of 100 mg/L. The biodegradation rate decreased significantly at lower and higher initial phenol concentrations of 50 and up to 3000 mg/L, reaching a rate of 0.1018/d. The results also indicate similar and up to double the degradation rate between the two bacterial states (encapsulated vs. suspended). High resolution scanning electron microscopy images of the SBP capsule's membrane morphology demonstrated a highly porous microfiltration membrane. These results, together with the long-term activity of the SBP capsules and verification that the culture remains pure after 60 days using 16S rRNA gene phylogenetic affiliation tests, provide evidence for a successful application of this new encapsulation technique for bioaugmentation of selected microbial cultures in water treatment processes.

A novel bioaugmentation treatment approach using a confined microbial environment: a case study in a Membrane Bioreactor wastewater treatment plant.

A novel bioaugmentation treatment approach, the Small-Bioreactor Platform (SBP) technology, was developed to increase the biological stabilization process in the treatment of wastewater in order to improve wastewater processing effectiveness. The SBP microfiltration membrane provides protection against the natural selection forces that target exogenous bacterial cultures within wastewater. As a result, the exogenous microorganisms culture adapt and proliferate, thus providing a successful bioaugmentation process in wastewater treatment. The new bioaugmentation treatment approach was studied in a full configuration Membrane Bioreactor (MBR) plant treating domestic wastewater. Our results present the potential of this innovative technology to eliminate, or reduce, the intensity of stress events, as well as shortening the recovery time after stress events, consequently elevating the treatment effectiveness. The effective dose of SBP capsules per cubic metre per day of wastewater was achieved during the addition of 3000 SBP capsules (1.25 SBP capsules per cubic metre per day), which provided approximately 4.5 L of high concentration exogenous biomass culture within the SBP capsules internal medium. This study demonstrates an innovative treatment capability which provides an effective bioaugmentation treatment in an MBR domestic wastewater treatment plant.

The potential of autochthonous microbial culture encapsulation in a confined environment for phenol biodegradation.

Olive mill wastewater (OMWW) is claimed to be one of the most polluting effluents produced by agro-food industries, providing high contaminants load that encase cytotoxic agents such as phenolic and polyphenolic compounds. Therefore, a significant and continuous stress episode is induced once the mixed liquor of the wastewater treatment plants (WWTP's) is being exposed to OMWW. The use of bio-augmentation treatment procedures can be useful to eliminate or reduce such stress episodes. In this study, we have estimated the use of autochthonous biomass implementation within small bioreactor platform (SBP) particles as a bio-augmentation method to challenge against WWTPs stress episodes. Our results showed that SBP particles significantly reduced the presence of various phenolics: tannic, gallic and caffeic acid in a synthetic medium and in crude OMWW matrix. Moreover, the SBP particles succeeded to biodegrade a very high concentration of phenol blend (3000 mg L(-1)). Our findings indicated that the presence of the SBP microfiltration membrane has reduced the phenol biodegradation rate by 50 % compared to the same suspended culture. Despite the observed reduction in biodegradation rate, encapsulation in a confined environment can offer significant values such as overcoming the grazing forcers and dilution, thus achieving a long-term sufficient biomass. The potential for reducing stress episodes caused by cytotoxic agents through bio-augmentation treatment procedure using the SBP technology is discussed.

Aminoglycosides affect intracellular Salmonella enterica serovars typhimurium and virchow.

The high antibacterial activity and selectivity of aminoglycosides and their low activity against intracellular bacteria associated with eukaryotic cells make them the antibiotics of choice for the elimination of extracellular bacteria during intracellular studies. Given the evidence that aminoglycosides can penetrate the eukaryotic cell membrane, the goal of this study was to examine the influence of aminoglycosides on macrophage-associated Salmonella. Herein, we show that gentamicin, kanamycin, and tobramycin at concentrations between 15 to 150 microg ml(-1) do not kill intracellular Salmonella but have other effects on the bacterial physiology. By using Salmonella enterica serovars Typhimurium and Virchow harboring luciferase reporter plasmid, we observed that the light produced by intracellular Salmonella declined immediately upon exposure to aminoglycosides, indicating that the bacteria were under stress. The extent of this effect was dependent on the macrophage host, on the identity of the aminoglycoside and its concentration, on the exposure time, and on the Salmonella serovar. Salmonella associated with Nramp1-negative macrophages, in which the phagosomal pH is higher, were more susceptible to aminoglycosides than Salmonella associated with Nramp1-expressing macrophages. These results verify that aminoglycosides affect intracellular bacteria and that the extent of this effect is dependent on the acidity level within the phagosome, suggesting that for the study of intracellular bacteria, the aminoglycoside concentration should be limited to two to five times the MIC for the bacterial strain studied. This precaution should guarantee the complete execution of extracellular bacteria with minimal effects on the intracellular bacteria and the host cells.

Expression of matrix metalloproteinases, inhibitor, and acid phosphatase in muscles of immobilized hindlimbs of rats.

External fixation procedures of limb immobilization provide excellent experimental models to study mechanisms involved in muscle disuse atrophy and recovery. Female Wistar rats (7-8 months old) had their right hindlimbs immobilized by an external fixation procedure for 5, 10, 21, and 30 days. Muscle mass of the gastrocnemius and quadriceps muscles was reduced by 41-46% in comparison with contralateral nonimmobilized legs. Acid phosphatase activities were significantly increased after 21 and 30 days of hindlimb immobilization. Histochemical staining for acid phosphatase activities increased in myofibers after the external fixation and also in macrophages in the adjacent extracellular matrix. Matrix metalloproteinase (MMP-2 and MMP-9) activities assessed by gel zymography and also a tissue inhibitor of metalloproteinases (TIMP-1) assessed by Western blot were elevated in the immobilized hindlimb muscles. Our study demonstrated that metalloproteinases are expressed relatively late after limb immobilization and appear to be responsible to a large degree for degradation of the extracellular matrix in experimental disuse atrophy.