Continuous UV-C/H2O2 and UV-C/Chlorine applied to municipal secondary effluent and nanofiltration retentate: Removal of contaminants of emerging concern, ecotoxicity, and reuse potential.

As global effects of water scarcity raise concerns and environmental regulations evolve, contemporary wastewater treatment plants (WWTPs) face the challenge of effectively removing a diverse range of contaminants of emerging concern (CECs) from municipal effluents. This study focuses on the assessment of advanced oxidation processes (AOPs), specifically UV-C/H2O2 and UV-C/Chlorine, for the removal of 14 target CECs in municipal secondary effluent (MSE, spiked with 10 µg L-1 of each CEC) or in the subsequent MSE nanofiltration retentate (NFR, no spiking). Phototreatments were carried out in continuous mode operation, with a hydraulic retention time of 3.4 min, using a tube-in-tube membrane photoreactor. For both wastewater matrices, UV-C photolysis (3.3 kJ L-1) exhibited high efficacy in removing CECs susceptible to photolysis, although lower treatment performance was observed for NFR. In MSE, adding 10 mg L-1 of H2O2 or Cl2 enhanced treatment efficiency, with UV-C/H2O2 outperforming UV-C/Chlorine. Both UV-C/AOPs eliminated the chronic toxicity of MSE toward Chlorella vulgaris. In the NFR, not only was the degradation of target CECs diminished, but chronic toxicity to C. vulgaris persisted after both UV-C/AOPs, with UV-C/Chlorine increasing toxicity due to potential toxic by-products. Nanofiltration permeate (NFP) exhibited low CECs and microbial content. A single chlorine addition effectively controlled Escherichia coli regrowth for 3 days, proving NFP potential for safe reuse in crop irrigation (<1 CFU/100 mL for E. coli; <1 mg L-1 for free chlorine). These findings provide valuable insights into the applications and limitations of UV-C/H2O2 and UV-C/Chlorine for distinct wastewater treatment scenarios.

A concept for international societally relevant microbiology education and microbiology knowledge promulgation in society.

EXECUTIVE SUMMARY: Microbes are all pervasive in their distribution and influence on the functioning and well-being of humans, life in general and the planet. Microbially-based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision-makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative-the IMiLI-is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence-based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner-centric, not academic microbiology-centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators-learners. As such, they will collect and analyse feedback from the end-users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators-learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships-a global societally relevant microbiology education ecosystem-in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. ABSTRACT: The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well-being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us-individuals/communities/nations/the human world-and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. HERE, WE PRESENT: our concept of how microbiology literacy may be achieved and the rationale underpinning it; the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision-making; and the key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner-centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091-1111). Importantly, although the primary target of the freely available (CC BY-NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity-driven, web-based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge.

Catalyst-free persulfate activation by UV/visible radiation for secondary urban wastewater disinfection.

This study focuses on the treatment of secondary urban wastewater (W) to improve the effluent quality aiming at the reduction of pathogenic microorganisms for the safe reuse of the treated wastewater (TW). Catalyst-free persulfate activation by radiation-based oxidation was applied as a treatment technology. A parametric study was carried out to select the best operating conditions. Total enterobacteria inactivation (quantified by the log reduction (CFU/100 mL)) was achieved when using [S2O82-] = 1 mM, pH = 8.5 (natural pH of W), T = 25 °C, and I = 500 W/m2. However, storing TW for 3 days promoted the regrowth of bacteria, risking its reutilization. Therefore, in this study, and for the first time, the potential beneficial role of inoculation of wastewater treated by the radiation-activated persulfate process with a diverse bacterial community was evaluated in order to control the regrowth of potentially harmful microorganisms through bacterial competition. For this, TW was diluted with river water (R) in the volume percentages of 5, 25, and 50 (percentages refer to R content), and enterobacteria and total heterotrophs were enumerated before and after storage for 72 h. The results showed total heterotrophs and enterobacteria regrowth for TW and R + TW diluted 5 and 25% after storage. However, for R + TW diluted 50%, only the total heterotrophs regrew. Hence, the treated wastewater generated by the oxidative process diluted with 50% river water complies with the legislated limits for reuse in urban uses or irrigation.

Disinfection of treated urban effluents for reuse by combination of coagulation/flocculation and Fenton processes.

In this study, a combination of coagulation/flocculation and Fenton processes was studied as tertiary treatment in order to generate treated water susceptible to reuse. The combination of both processes has never been applied in disinfection of real urban wastewater. The best removals of turbidity and enterobacteria were achieved when applying a coagulant (FeCl3) dosage of 120 mg/L and the natural pH of the effluent (7.14). The following Fenton reaction presented the maximal enterobacteria inactivation after 120 min at 25 °C, when using hydrogen peroxide and added iron concentrations of 100 mg/L and 7 mg/L, respectively. The abundance of antibiotic resistant (amoxicillin and sulfamethoxazole) enterobacteria and total enterobacteria, enterococci, and heterotrophs, and antibiotic resistance genes - ARG - (sul1, blaTEM and qnrS) was evaluated before and after each step of the treatment. Values below 10 CFU/100 mL were achieved for total and resistant cultivable enterobacteria immediately after treatment and after storage for 72 h, therefore meeting the strictest limit imposed for E. coli. Physico-chemical parameters also met the established limits for water reuse. Despite harbouring a rich and diverse bacterial community, the final stored disinfected wastewater contained high relative abundance of potentially hazardous bacteria. Such results point out the need of a deep microbiological characterization of treated wastewater to evaluate the risk of its reuse in irrigation.

Application of iron-activated persulfate for municipal wastewater disinfection.

To address the increasing contamination of aquatic environments and incidence of waterborne diseases, advanced oxidation processes with activated persulfate have emerged as tools to inactivate wastewater microorganisms and contaminants. In this work, the disinfection of a secondary effluent from a wastewater treatment plant by iron-based persulfate activation was studied. Experiments in a batch stirred tank reactor were carried out to evaluate the performance along reaction time and the effect of operational parameters in the oxidative process efficiency (oxidant and iron concentration, pH and temperature). After 60 min of reaction, persulfate and iron concentrations of 3 mM and 0.75 mM, respectively, combined with a neutral initial pH (7.5) and a temperature of 40 °C, allowed to reach values below the detection limit (<10 CFU/100 mL) of enterococci and enterobacteria with and without ciprofloxacin resistance, as well as a 91% inactivation of total heterotrophic organisms and a 70% removal of total organic carbon. Regrowth of microorganisms was evaluated 72 h after treatment and it was only noticed a slight increase in total heterotrophs. Evaluation of physico-chemical characteristics of the treated water showed that it meets the requirements imposed by European and Portuguese legislation for its reuse in irrigation and most urban utilities.

Polyphasic characterization of carbapenem-resistant Klebsiella pneumoniae clinical isolates suggests vertical transmission of the blaKPC-3 gene.

Carbapenem-resistant Klebsiella pneumoniae are a major global threat in healthcare facilities. The propagation of carbapenem resistance determinants can occur through vertical transmission, with genetic elements being transmitted by the host bacterium, or by horizontal transmission, with the same genetic elements being transferred among distinct bacterial hosts. This work aimed to track carbapenem resistance transmission by K. pneumoniae in a healthcare facility. The study involved a polyphasic approach based on conjugation assays, resistance phenotype and genotype analyses, whole genome sequencing, and plasmid characterization by pulsed field gel electrophoresis and optical DNA mapping. Out of 40 K. pneumoniae clinical isolates recovered over two years, five were carbapenem- and multidrug-resistant and belonged to multilocus sequence type ST147. These isolates harboured the carbapenemase encoding blaKPC-3 gene, integrated in conjugative plasmids of 140 kbp or 55 kbp, belonging to replicon types incFIA/incFIIK or incN/incFIIK, respectively. The two distinct plasmids encoding the blaKPC-3 gene were associated with distinct genetic lineages, as confirmed by optical DNA mapping and whole genome sequence analyses. These results suggested vertical (bacterial strain-based) transmission of the carbapenem-resistance genetic elements. Determination of the mode of transmission of antibiotic resistance in healthcare facilities, only possible based on polyphasic approaches as described here, is essential to control resistance propagation.

Visualizing the invisible: class excursions to ignite children's enthusiasm for microbes.

We have recently argued that, because microbes have pervasive - often vital - influences on our lives, and that therefore their roles must be taken into account in many of the decisions we face, society must become microbiology-literate, through the introduction of relevant microbiology topics in school curricula (Timmis et al. 2019. Environ Microbiol 21: 1513-1528). The current coronavirus pandemic is a stark example of why microbiology literacy is such a crucial enabler of informed policy decisions, particularly those involving preparedness of public-health systems for disease outbreaks and pandemics. However, a significant barrier to attaining widespread appreciation of microbial contributions to our well-being and that of the planet is the fact that microbes are seldom visible: most people are only peripherally aware of them, except when they fall ill with an infection. And it is disease, rather than all of the positive activities mediated by microbes, that colours public perception of 'germs' and endows them with their poor image. It is imperative to render microbes visible, to give them life and form for children (and adults), and to counter prevalent misconceptions, through exposure to imagination-capturing images of microbes and examples of their beneficial outputs, accompanied by a balanced narrative. This will engender automatic mental associations between everyday information inputs, as well as visual, olfactory and tactile experiences, on the one hand, and the responsible microbes/microbial communities, on the other hand. Such associations, in turn, will promote awareness of microbes and of the many positive and vital consequences of their actions, and facilitate and encourage incorporation of such consequences into relevant decision-making processes. While teaching microbiology topics in primary and secondary school is key to this objective, a strategic programme to expose children directly and personally to natural and managed microbial processes, and the results of their actions, through carefully planned class excursions to local venues, can be instrumental in bringing microbes to life for children and, collaterally, their families. In order to encourage the embedding of microbiology-centric class excursions in current curricula, we suggest and illustrate here some possibilities relating to the topics of food (a favourite pre-occupation of most children), agriculture (together with horticulture and aquaculture), health and medicine, the environment and biotechnology. And, although not all of the microbially relevant infrastructure will be within reach of schools, there is usually access to a market, local food store, wastewater treatment plant, farm, surface water body, etc., all of which can provide opportunities to explore microbiology in action. If children sometimes consider the present to be mundane, even boring, they are usually excited with both the past and the future so, where possible, visits to local museums (the past) and research institutions advancing knowledge frontiers (the future) are strongly recommended, as is a tapping into the natural enthusiasm of local researchers to leverage the educational value of excursions and virtual excursions. Children are also fascinated by the unknown, so, paradoxically, the invisibility of microbes makes them especially fascinating objects for visualization and exploration. In outlining some of the options for microbiology excursions, providing suggestions for discussion topics and considering their educational value, we strive to extend the vistas of current class excursions and to: (i) inspire teachers and school managers to incorporate more microbiology excursions into curricula; (ii) encourage microbiologists to support school excursions and generally get involved in bringing microbes to life for children; (iii) urge leaders of organizations (biopharma, food industries, universities, etc.) to give school outreach activities a more prominent place in their mission portfolios, and (iv) convey to policymakers the benefits of providing schools with funds, materials and flexibility for educational endeavours beyond the classroom.

Ozonation and UV254nm radiation for the removal of microorganisms and antibiotic resistance genes from urban wastewater.

Conventional wastewater treatment has a limited capacity to reduce antibiotic resistant bacteria and genes (ARB&ARG). Tertiary treatment processes are promising solutions, although the transitory inactivation of bacteria may select ARB&ARG. This study aimed at assessing the potential of ozonation and UV254nm radiation to inactivate cultivable fungal and bacterial populations, and the selected genes 16S rRNA (common to all bacteria), intI1 (common in Gram-negative bacteria) and the ARG vanA, blaTEM, sul1 and qnrS. The abundance of the different microbiological parameters per volume of wastewater was reduced by ∼2 log units for cultivable fungi and 16S rRNA and intI1 genes, by∼3-4 log units, for total heterotrophs, enterobacteria and enterococci, and to values close or below the limits of quantification for ARG, for both processes, after a contact time of 30min. Yet, most of the cultivable populations, the 16S rRNA and intI1 genes as well as the ARG, except qnrS after ozonation, reached pre-treatment levels after 3days storage, suggesting a transitory rather than permanent microbial inactivation. Noticeably, normalization per 16S rRNA gene evidenced an increase of the ARG and intI1 prevalence, mainly after UV254nm treatment. The results suggest that these tertiary treatments may be selecting for ARB&ARG populations.

Structure-guided engineering of molinate hydrolase for the degradation of thiocarbamate pesticides.

Molinate is a recalcitrant thiocarbamate used to control grass weeds in rice fields. The recently described molinate hydrolase, from Gulosibacter molinativorax ON4T, plays a key role in the only known molinate degradation pathway ending in the formation of innocuous compounds. Here we report the crystal structure of recombinant molinate hydrolase at 2.27 Å. The structure reveals a homotetramer with a single mononuclear metal-dependent active site per monomer. The active site architecture shows similarities with other amidohydrolases and enables us to propose a general acid-base catalysis mechanism for molinate hydrolysis. Molinate hydrolase is unable to degrade bulkier thiocarbamate pesticides such as thiobencarb which is used mostly in rice crops. Using a structural-based approach, we were able to generate a mutant (Arg187Ala) that efficiently degrades thiobencarb. The engineered enzyme is suitable for the development of a broader thiocarbamate bioremediation system.

Molinate quantification in environmental water by a glutathione-S-transferase based biosensor.

A glutathione-S-transferase (GST) based biosensor was developed to quantify the thiocarbamate herbicide molinate in environmental water. The biosensor construction was based on GST immobilization onto a glassy carbon electrode via aminosilane-glutaraldehyde covalent attachment. The principle supporting the use of this biosensor consists of the GST inhibition process promoted by molinate. Differential pulse voltammetry was used to obtain a calibration curve for molinate concentration, ranging from 0.19 to 7.9 mg L(-1) and presenting a detection limit of 0.064 mg L(-1). The developed biosensor is stable, and reusable during 15 days. The GST-based biosensor was successfully applied to quantify molinate in rice paddy field floodwater samples. The results achieved with the developed biosensor were in accordance with those obtained by high performance liquid chromatography. The proposed device is suitable for screening environmental water analysis and, since no sample preparation is required, it can be used in situ and in real-time measurements.

Acinetobacter rudis sp. nov., isolated from raw milk and raw wastewater.

Two bacterial strains, G30(T) and A1PC16, isolated respectively from raw milk and raw wastewater, were characterized using a polyphasic approach. Chemotaxonomic characterization supported the inclusion of these strains in the genus Acinetobacter, with Q-8 and Q-9 as the major respiratory quinones, genomic DNA G+C contents within the range observed for this genus (38-47 mol%) and C(16:0), C(18:1)ω9c and C(16:1)ω7c/iso-C(15:0) 2-OH as the predominant fatty acids. The observation of 16S rRNA gene sequence similarity lower than 97% with other Acinetobacter species with validly published names led to the hypothesis that these isolates could represent a novel species. This hypothesis was supported by comparative analysis of partial sequences of the genes rpoB and gyrB, which showed that strains G30(T) and A1PC16 did not cluster with any species with validly published names, forming a distinct lineage. DNA-DNA hybridizations confirmed that the two strains were members of the same species, which could be distinguished from their congeners by several phenotypic characteristics. On the basis of these arguments, it is proposed that strains G30(T) and A1PC16 represent a novel species, for which the name Acinetobacter rudis sp. nov. is proposed. The type strain is strain G30(T) (=LMG 26107(T) =CCUG 57889(T) =DSM 24031(T) =CECT 7818(T)).

Candidimonas nitroreducens gen. nov., sp. nov. and Candidimonas humi sp. nov., isolated from sewage sludge compost.

Two bacterial strains (SC-089(T) and SC-092(T)) isolated from sewage sludge compost were characterized by using a polyphasic approach. The isolates were Gram-negative short rods, catalase- and oxidase-positive, and showed good growth at 30 °C, at pH 7 and with 1 % (w/v) NaCl. Ubiquinone 8 was the major respiratory quinone, and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol were amongst the major polar lipids. On the basis of 16S rRNA gene sequence analysis, the strains were observed to be members of the family Alcaligenaceae, but could not be identified as members of any validly described genus. The low levels of 16S rRNA gene sequence similarity to other recognized taxa, together with comparative analysis of phenotypic traits and chemotaxonomic markers, supported the proposal of a new genus within the family Alcaligenaceae, for which the name Candidimonas gen. nov. is proposed. Strains SC-089(T) and SC-092(T), which shared 99.1 % 16S rRNA gene sequence similarity, could be differentiated at the phenotypic level, and DNA-DNA hybridization results supported their identification as representing distinct species. The names proposed for these novel species are Candidimonas nitroreducens sp. nov. (type strain, SC-089(T) = LMG 24812(T) = CCUG 55806(T)) and Candidimonas humi sp. nov. (type strain, SC-092(T) = LMG 24813(T) = CCUG 55807(T)).

Paenibacillus residui sp. nov., isolated from urban waste compost.

Two bacterial strains, MC-246(T) and MC-247, were isolated from municipal urban waste compost and characterized by a polyphasic approach. Both isolates were Gram-stain-variable, endospore-forming rods that were catalase-, oxidase- and ß-galactosidase-positive, and able to grow at 25-50°C and pH 7.0-9.0, with optimum growth at 37°C and pH 7. The predominant cellular fatty acids were anteiso-C15:0, iso-C15:0, iso-C16: 0, anteiso-C17:0 and iso-C17:0; the major respiratory quinone was menaquinone MK-7; the cell wall peptidoglycan was of type A1γ; and the DNA G+C content was 49 mol%. These characteristics, as well as data from 16S RNA gene sequence analysis, showed that these strains were affiliated with the genus Paenibacillus; the type strains of Paenibacillus ginsengarvi and Paenibacillus hodogayensis were among their closest neighbours (< 94.2 % sequence similarity). Nevertheless, the hypothesis that strains MC-246(T) and MC-247 could represent a novel species was supported by the low 16S rRNA gene sequence similarity values shared with other members of the genus Paenibacillus and by the observation of distinct biochemical and physiological traits. Strains MC-246(T) and MC-247 shared 99.6 % 16S rRNA gene sequence similarity and showed almost identical MALDI-TOF mass spectra, but could be distinguished at the phenotypic and genotypic level. However, DNA-DNA hybridization between strains MC-246(T) and MC-247 resulted in values above 70 % indicating that both organisms represent a single species, for which the name Paenibacillus residui sp. nov. is proposed; the type strain is MC-246(T) (=DSM 22072(T) =CCUG 57263(T)).

Shinella fusca sp. nov., isolated from domestic waste compost.

A bacterium, designated strain DC-196(T), isolated from kitchen refuse compost was analysed by using a polyphasic approach. Strain DC-196(T) was characterized as a Gram-negative short rod that was catalase- and oxidase-positive, and able to grow at 10-40 degrees C, pH 6-9 and in NaCl concentrations as high as 3 %. Chemotaxonomically, C(18 : 1) was observed to be the predominant cellular fatty acid and ubiquinone 10 (Q10) was the predominant respiratory quinone. The G+C content of the genomic DNA was determined to be 66 mol%. On the basis of the genotypic, phenotypic and chemotaxonomic characteristics, strain DC-196(T) was assigned to the genus Shinella, although with distinctive features. At the time of writing, 16S rRNA gene sequence similarities of 97.6-96.8 % and the low DNA-DNA hybridization values of 38.2-32.2 % with the type strains of the three recognized Shinella species confirmed that strain DC-196(T) represents a novel species of the genus, for which the name Shinella fusca sp. nov. is proposed (type strain DC-196(T)=CCUG 55808(T)=LMG 24714(T)).

Microbacterium invictum sp. nov., isolated from homemade compost.

Strain DC-200T was isolated from homemade compost produced from kitchen refuse and characterized using a polyphasic approach. The isolate was a Gram-positive motile short rod, facultatively aerobic, catalase-positive and oxidase-negative, and was able to grow at 10-37 degrees C, pH 6.0-9.5 and with up to 5% of NaCl. The peptidoglycan was of the type B1 alpha and the muramic acid residues were glycolylated. The major fatty acids were anteiso-C15:0 and anteiso-C17:0. The predominant respiratory menaquinones were MK-11 and MK-12. The G+C content of the genomic DNA was 70 mol%. Based on the analysis of the 16S rRNA gene sequence, the closest phylogenetic neighbours of strain DC-200T were Microbacterium lacus A5E-52T (98.7%) and Microbacterium aoyamense KV-492T (98.2%). The phenetic characterization of the isolate supports its inclusion within the genus Microbacterium; however, its distinctive phenotypic features and the results from the 16S rRNA gene sequence analysis and the DNA-DNA hybridization study suggest that the isolate represents a novel species. The name Microbacterium invictum sp. nov. is proposed. The type strain is DC-200T (=DSM 19600T=LMG 24557T).

Treatment of cork boiling wastewater using chemical oxidation and biodegradation.

Three cultures were enriched from cork boiling wastewater using tannic acid as the selective carbon substrate, at 25 degrees C and pH 7.2, 25 degrees C and pH 4.7 and 50 degrees C and pH 4.7. The enrichment culture obtained at neutral pH was composed of five culturable isolates, whereas from each acidic enrichment two bacterial strains were isolated. Mesophilic isolates were Gram negative bacteria belonging to the genera Klebsiella, Pseudomonas, Stenotrophomonas and Burkholderia. Thermophilic isolates were members of the genus Bacillus. Despite the capability of the enrichment cultures to use tannic acid as single carbon and energy source, those cultures were unable to reduce the total polyphenols or the total organic carbon content of cork boiling wastewater. In order to increase the bioavailability of the organic carbon in cork boiling wastewater, biodegradation was preceded by Fenton oxidation. It was demonstrated that the combined process, using small amounts of Fenton reagents and biodegradative inoculum added almost simultaneously to cork boiling wastewater, leads to TOC reductions of more than 90%.