The nutritional composition and cell size of microbial biomass for food applications are defined by the growth conditions.

BACKGROUND: It is increasingly recognized that conventional food production systems are not able to meet the globally increasing protein needs, resulting in overexploitation and depletion of resources, and environmental degradation. In this context, microbial biomass has emerged as a promising sustainable protein alternative. Nevertheless, often no consideration is given on the fact that the cultivation conditions affect the composition of microbial cells, and hence their quality and nutritional value. Apart from the properties and nutritional quality of the produced microbial food (ingredient), this can also impact its sustainability. To qualitatively assess these aspects, here, we investigated the link between substrate availability, growth rate, cell composition and size of Cupriavidus necator and Komagataella phaffii. RESULTS: Biomass with decreased nucleic acid and increased protein content was produced at low growth rates. Conversely, high rates resulted in larger cells, which could enable more efficient biomass harvesting. The proteome allocation varied across the different growth rates, with more ribosomal proteins at higher rates, which could potentially affect the techno-functional properties of the biomass. Considering the distinct amino acid profiles established for the different cellular components, variations in their abundance impacts the product quality leading to higher cysteine and phenylalanine content at low growth rates. Therefore, we hint that costly external amino acid supplementations that are often required to meet the nutritional needs could be avoided by carefully applying conditions that enable targeted growth rates. CONCLUSION: In summary, we demonstrate tradeoffs between nutritional quality and production rate, and we discuss the microbial biomass properties that vary according to the growth conditions.

PrsQ2, a small periplasmic protein involved in increased uranium resistance in the bacterium Cupriavidus metallidurans.

Uranium contamination is a widespread problem caused by natural and anthropogenic activities. Although microorganisms thrive in uranium-contaminated environments, little is known about the actual molecular mechanisms mediating uranium resistance. Here, we investigated the resistance mechanisms driving the adaptation of Cupriavidus metallidurans NA4 to toxic uranium concentrations. We selected a spontaneous mutant able to grow in the presence of 1 mM uranyl nitrate compared to 250 µM for the parental strain. The increased uranium resistance was acquired via the formation of periplasmic uranium-phosphate precipitates facilitated by the increased expression of a genus-specific small periplasmic protein, PrsQ2, regulated as non-cognate target of the CzcS2-CzcR2 two-component system. This study shows that bacteria can adapt to toxic uranium concentrations and explicates the complete genetic circuit behind the adaptation.

Cupriavidus metallidurans NA4 actively forms polyhydroxybutyrate-associated uranium-phosphate precipitates.

Cupriavidus metallidurans is a model bacterium to study molecular metal resistance mechanisms and its use for the bioremediation of several metals has been shown. However, its mechanisms for radionuclide resistance are unexplored. We investigated the interaction with uranium and associated cellular response to uranium for Cupriavidus metallidurans NA4. Strain NA4 actively captured 98 ± 1% of the uranium in its biomass after growing 24 h in the presence of 100 µM uranyl nitrate. TEM HAADF-EDX microscopy confirmed intracellular uranium-phosphate precipitates that were mainly associated with polyhydroxybutyrate. Furthermore, whole transcriptome sequencing indicated a complex transcriptional response with upregulation of genes encoding general stress-related proteins and several genes involved in metal resistance. More in particular, gene clusters known to be involved in copper and silver resistance were differentially expressed. This study provides further insights into bacterial interactions with and their response to uranium. Our results could be promising for uranium bioremediation purposes with the multi-metal resistant bacterium C. metallidurans NA4.

Effective orthophosphate removal from surface water using hydrogen-oxidizing bacteria: Moving towards applicability.

Effective orthophosphate removal strategies are needed to counteract eutrophication and guarantee water quality. Previously, we established that hydrogen-oxidizing bacteria (HOB) have the ability to remove orthophosphate from artificial surface water. In the present study, we expand the application of the HOB orthophosphate removal strategy (1) to treat artificial surface water with low initial orthophosphate concentrations, (2) to treat real surface water and real wastewater effluent, and (3) to remove orthophosphate continuously. For synthetic surface water, irrespective of the initial concentration of 0.7, 0.5, 0.3, and 0.1 mg PO43--P/L, ultra-low concentrations (0.0058 ± 0.0028 mg PO43--P/L) were obtained. When artificial surface water was replaced by real surface water, without added nutrients or other chemicals, it was shown that over 90% orthophosphate could be removed within 30 min of operation in a batch configuration (0.031 ± 0.023 mg PO43--P/L). In continuous operation, orthophosphate removal from surface water left an average concentration of 0.040 ± 0.036 for 60 days, and the lowest orthophosphate concentration measured was 0.013 mg PO43-/L. Simultaneously, nitrate was continuously removed for 60 days below 0.1 mg/L. The ability to remove orthophosphate even under nitrogen limiting conditions might be related to the ability of HOB to fix nitrogen. This study brings valuable insights into the potential use of HOB biofilms for nutrient remediation and recovery.

In-Depth Observation on the Microbial and Fungal Community Structure of Four Contrasting Tomato Cultivation Systems in Soil Based and Soilless Culture Systems.

As soil and soilless culture systems are highly dynamic environments, the structure of rhizosphere microbial communities is consistently adapting. There is a knowledge gap between the microbial community structure of soil based and soilless culture systems and thus we aimed at surveying their impact on diversity and composition of bacterial communities across a 10-month period in a tomato cultivation system. We compared community metrics between an soil based culture system fertilized with malt sprouts and blood meal, known for its slow and high mineralization rate, respectively and a soilless culture system fertilized with fish effluent or supplemented with an liquid organic fertilizer. Bacterial and fungal community composition was followed over time using two complementary techniques, phospholipid fatty acid analysis and 16S rRNA amplicon sequencing. Nitrogen dynamics and plant performance were assessed to provide insight on how bacterial diversity of soil and soilless microbial communities ultimately impacts productivity. Similar plant performance was observed in soilless culture systems and soil based system and yield was the highest with the aquaponics-derived fertilizer. Soil and soilless cultivating systems supplemented with different nitrogen-rich fertilizers differed on its characteristics throughout the experimental period. Fast-paced fluctuations in pH(H2O) and nutrient cycling processes were observed in growing medium. Physicochemical characteristics changed over time and interacted with bacterial community metrics. Multivariate analysis showed that plant length, pH, Flavisolibacter, phosphorus, chloride, ammonium, potassium, calcium, magnesium, sodium, electrical conductivity, nitrate, sulfate, and the bacterial genera Desulfotomaculum, Solirubrobacter, Dehalococcoides, Bythopirellula, Steroidobacter, Litorilinea, Nonomuraea were the most significant factors discriminating between natural soils supplemented with animal and plant by-products. Long-term fertilizer regimes significantly changed the PLFA fingerprints in both the soilless culture and soil based culture system. The use of these by-products in the soil was positively associated with arbuscular mycorrhizal fungi (AMF), which may influence rhizosphere communities through root exudates and C translocation. Community structure was distinct and consistently different over time, despite the fertilizer supplementation. The fungal microbial community composition was less affected by pH, while the composition of the bacterial communities (Actinomycetes, Gram-negative bacteria, and Gram-positive bacteria) was closely defined by soil pH, demonstrating the significance of pH as driver of bacterial community composition. Fertilizer application may be responsible for variations over time in the ecosystem. Knowledge about the microbial interactions in tomato cultivating systems opens a window of opportunity for designing targeted fertilizers supporting sustainable crop production.

Hydrogen oxidizing bacteria are capable of removing orthophosphate to ultra-low concentrations in a fed batch reactor configuration.

This paper proposes the use of hydrogen oxidizing bacteria (HOB) for the removal of orthophosphate from surface water as treatment step to prevent cyanobacterial blooms. To be effective as an orthophosphate removal strategy, an efficient transfer of hydrogen to the HOB is essential. A trickling filter was selected for this purpose. Using this system, a removal rate of 11.32 ± 0.43 mg PO4-3-P/L.d was achieved. The HOB biomass, developed on the trickling filter, is composed of 1.25% phosphorus on dry matter, which suggests that the orthophosphate removal principle is based on HOB growth. Cyanobacterial growth assays of the untreated and treated water showed that Synechocystis sp was only able to grow in the untreated water. Orthophosphate was removed to average residual values of 0.008 mg/L. In this proof of principle study, it is shown that HOB are able to remove orthophosphate from water to concentrations that prevent cyanobacterial growth.

Microbial enrichment, functional characterization and isolation from a cold seep yield piezotolerant obligate hydrocarbon degraders.

Deep-sea environments can become contaminated with petroleum hydrocarbons. The effects of hydrostatic pressure (HP) in the deep sea on microbial oil degradation are poorly understood. Here, we performed long-term enrichments (100 days) from a natural cold seep while providing optimal conditions to sustain high hydrocarbon degradation rates. Through enrichments performed at increased HP and ambient pressure (AP) and by using control enrichments with marine broth, we demonstrated that both pressure and carbon source can have a big impact on the community structure. In contrast to previous studies, hydrocarbonoclastic operational taxonomic units (OTUs) remained dominant at both AP and increased HP, suggesting piezotolerance of these OTUs over the tested pressure range. Twenty-three isolates were obtained after isolation and dereplication. After recultivation at increased HP, an Alcanivorax sp. showed promising piezotolerance in axenic culture. Furthermore, preliminary co-cultivation tests indicated synergistic growth between some isolates, which shows promise for future synthetic community construction. Overall, more insights into the effect of increased HP on oil-degrading communities were obtained as well as several interesting isolates, e.g. a piezotolerant hydrocarbonoclastic bacterium for future deep-sea bioaugmentation investigation.

Gene Expansion and Positive Selection as Bacterial Adaptations to Oligotrophic Conditions.

We examined the genomic adaptations of prevalent bacterial taxa in a highly nutrient- and ion-depleted freshwater environment located in the secondary cooling water system of a nuclear research reactor. Using genome-centric metagenomics, we found that none of the prevalent bacterial taxa were related to typical freshwater bacterial lineages. We also did not identify strong signatures of genome streamlining, which has been shown to be one of the ecoevolutionary forces shaping the genome characteristics of bacterial taxa in nutrient-depleted environments. Instead, focusing on the dominant taxon, a novel Ramlibacter sp. which we propose to name Ramlibacter aquaticus, we detected extensive positive selection on genes involved in phosphorus and carbon scavenging pathways. These genes were involved in the high-affinity phosphate uptake and storage into polyphosphate granules, metabolism of nitrogen-rich organic matter, and carbon/energy storage into polyhydroxyalkanoate. In parallel, comparative genomics revealed a high number of paralogs and an accessory genome significantly enriched in environmental sensing pathways (i.e., chemotaxis and motility), suggesting extensive gene expansions in R. aquaticus The type strain of R. aquaticus (LMG 30558T) displayed optimal growth kinetics and productivity at low nutrient concentrations, as well as substantial cell size plasticity. Our findings with R. aquaticus LMG 30558T demonstrate that positive selection and gene expansions may represent successful adaptive strategies to oligotrophic environments that preserve high growth rates and cellular productivity.IMPORTANCE By combining a genome-centric metagenomic approach with a culture-based approach, we investigated the genomic adaptations of prevalent populations in an engineered oligotrophic freshwater system. We found evidence for widespread positive selection on genes involved in phosphorus and carbon scavenging pathways and for gene expansions in motility and environmental sensing to be important genomic adaptations of the abundant taxon in this system. In addition, microscopic and flow cytometric analysis of the first freshwater representative of this population (Ramlibacter aquaticus LMG 30558T) demonstrated phenotypic plasticity, possibly due to the metabolic versatility granted by its larger genome, to be a strategy to cope with nutrient limitation. Our study clearly demonstrates the need for the use of a broad set of genomic tools combined with culture-based physiological characterization assays to investigate and validate genomic adaptations.

Reduced TCA cycle rates at high hydrostatic pressure hinder hydrocarbon degradation and obligate oil degraders in natural, deep-sea microbial communities.

Petroleum hydrocarbons reach the deep-sea following natural and anthropogenic factors. The process by which they enter deep-sea microbial food webs and impact the biogeochemical cycling of carbon and other elements is unclear. Hydrostatic pressure (HP) is a distinctive parameter of the deep sea, although rarely investigated. Whether HP alone affects the assembly and activity of oil-degrading communities remains to be resolved. Here we have demonstrated that hydrocarbon degradation in deep-sea microbial communities is lower at native HP (10 MPa, about 1000 m below sea surface level) than at ambient pressure. In long-term enrichments, increased HP selectively inhibited obligate hydrocarbon-degraders and downregulated the expression of beta-oxidation-related proteins (i.e., the main hydrocarbon-degradation pathway) resulting in low cell growth and CO2 production. Short-term experiments with HP-adapted synthetic communities confirmed this data, revealing a HP-dependent accumulation of citrate and dihydroxyacetone. Citrate accumulation suggests rates of aerobic oxidation of fatty acids in the TCA cycle were reduced. Dihydroxyacetone is connected to citrate through glycerol metabolism and glycolysis, both upregulated with increased HP. High degradation rates by obligate hydrocarbon-degraders may thus be unfavourable at increased HP, explaining their selective suppression. Through lab-scale cultivation, the present study is the first to highlight a link between impaired cell metabolism and microbial community assembly in hydrocarbon degradation at high HP. Overall, this data indicate that hydrocarbons fate differs substantially in surface waters as compared to deep-sea environments, with in situ low temperature and limited nutrients availability expected to further prolong hydrocarbons persistence at deep sea.

Butyrate-producing bacteria supplemented in vitro to Crohn's disease patient microbiota increased butyrate production and enhanced intestinal epithelial barrier integrity.

The management of the dysbiosed gut microbiota in inflammatory bowel diseases (IBD) is gaining more attention as a novel target to control this disease. Probiotic treatment with butyrate-producing bacteria has therapeutic potential since these bacteria are depleted in IBD patients and butyrate has beneficial effects on epithelial barrier function and overall gut health. However, studies assessing the effect of probiotic supplementation on microbe-microbe and host-microbe interactions are rare. In this study, butyrate-producing bacteria (three mono-species and one multispecies mix) were supplemented to the fecal microbial communities of ten Crohn's disease (CD) patients in an in vitro system simulating the mucus- and lumen-associated microbiota. Effects of supplementation in short-chain fatty acid levels, bacterial colonization of mucus environment and intestinal epithelial barrier function were evaluated. Treatment with F. prausnitzii and the mix of six butyrate-producers significantly increased the butyrate production by 5-11 mol%, and colonization capacity in mucus- and lumen-associated CD microbiota. Treatments with B. pullicaecorum 25-3T and the mix of six butyrate-producers improved epithelial barrier integrity in vitro. This study provides proof-of-concept data for the therapeutic potential of butyrate-producing bacteria in CD and supports the future preclinical development of a probiotic product containing butyrate-producing species.

The Impact of Space Flight on Survival and Interaction of Cupriavidus metallidurans CH34 with Basalt, a Volcanic Moon Analog Rock.

Microbe-mineral interactions have become of interest for space exploration as microorganisms could be used to biomine from extra-terrestrial material and extract elements useful as micronutrients in life support systems. This research aimed to identify the impact of space flight on the long-term survival of Cupriavidus metallidurans CH34 in mineral water and the interaction with basalt, a lunar-type rock in preparation for the ESA spaceflight experiment, BIOROCK. Therefore, C. metallidurans CH34 cells were suspended in mineral water supplemented with or without crushed basalt and send for 3 months on board the Russian FOTON-M4 capsule. Long-term storage had a significant impact on cell physiology and energy status (by flow cytometry analysis, plate count and intracellular ATP measurements) as 60% of cells stored on ground lost their cell membrane potential, only 17% were still active, average ATP levels per cell were significantly lower and cultivability dropped to 1%. The cells stored in the presence of basalt and exposed to space flight conditions during storage however showed less dramatic changes in physiology, with only 16% of the cells lost their cell membrane potential and 24% were still active, leading to a higher cultivability (50%) and indicating a general positive effect of basalt and space flight on survival. Microbe-mineral interactions and biofilm formation was altered by spaceflight as less biofilm was formed on the basalt during flight conditions. Leaching from basalt also changed (measured with ICP-OES), showing that cells release more copper from basalt and the presence of cells also impacted iron and magnesium concentration irrespective of the presence of basalt. The flight conditions thus could counteract some of the detrimental effects observed after the 3 month storage conditions.

Microalgal bacterial flocs treating paper mill effluent: A sunlight-based approach for removing carbon, nitrogen, phosphorus, and calcium.

Treatment of upflow anaerobic sludge blanket (UASB) effluent from a paper mill in aerated activated sludge reactors involves high aeration costs. Moreover, this calcium-rich effluent leads to problematic scale formation. Therefore, a novel strategy for the aerobic treatment of paper mill UASB effluent in microalgal bacterial floc sequencing batch reactors (MaB-floc SBRs) is proposed, in which oxygen is provided via photosynthesis, and calcium is removed via bio-mineralization. Based on the results of batch experiments in the course of this study, a MaB-floc SBR was operated at an initial neutral pH. This SBR removed 58±21% organic carbon, 27±8% inorganic carbon, 77±5% nitrogen, 73±2% phosphorus, and 27±11% calcium. MaB-flocs contained 10±3% calcium, including biologically-influenced calcite crystals. The removal of calcium and inorganic carbon by MaB-flocs significantly decreased when inhibiting extracellular carbonic anhydrase (CA), an enzyme that catalyses the hydration and dehydration of CO2. This study demonstrates the potential of MaB-floc SBRs for the alternative treatment of calcium-rich paper mill effluent, and highlights the importance of extracellular CA in this treatment process.

Bacillus sphaericus LMG 22257 is physiologically suitable for self-healing concrete.

The suitability of using a spore-forming ureolytic strain, Bacillus sphaericus, was evaluated for self-healing of concrete cracks. The main focus was on alkaline tolerance, calcium tolerance, oxygen dependence, and low-temperature adaptability. Experimental results show that B. sphaericus had a good tolerance. It can grow and germinate in a broad range of alkaline pH. The optimal pH range is 7 âˆ¼ 9. High alkaline conditions (pH 10 âˆ¼ 11) slow down but not stop the growth and germination. Oxygen was strictly needed during bacterial growth and germination, but not an essential factor during bacterial urea decomposition. B. sphaericus also had a good Ca tolerance, especially at a high bacterial concentration of 108 cells/mL; no significant influence was observed on bacterial ureolytic activity of the presence of 0.9M Ca2+. Furthermore, at a low temperature (10 °C), bacterial spores germinated and revived ureolytic activity with some retardation. However, this retardation can be counteracted by using a higher bacterial concentration and by supplementing yeast extract. It can be concluded that B. sphaericus is a suitable bacterium for application in bacteria-based self-healing concrete.

Microbial oil-degradation under mild hydrostatic pressure (10 MPa): which pathways are impacted in piezosensitive hydrocarbonoclastic bacteria?

Oil spills represent an overwhelming carbon input to the marine environment that immediately impacts the sea surface ecosystem. Microbial communities degrading the oil fraction that eventually sinks to the seafloor must also deal with hydrostatic pressure, which linearly increases with depth. Piezosensitive hydrocarbonoclastic bacteria are ideal candidates to elucidate impaired pathways following oil spills at low depth. In the present paper, we tested two strains of the ubiquitous Alcanivorax genus, namely A. jadensis KS_339 and A. dieselolei KS_293, which is known to rapidly grow after oil spills. Strains were subjected to atmospheric and mild pressure (0.1, 5 and 10 MPa, corresponding to a depth of 0, 500 and 1000 m, respectively) providing n-dodecane as sole carbon source. Pressures equal to 5 and 10 MPa significantly lowered growth yields of both strains. However, in strain KS_293 grown at 10 MPa CO2 production per cell was not affected, cell integrity was preserved and PO4(3-) uptake increased. Analysis of its transcriptome revealed that 95% of its genes were downregulated. Increased transcription involved protein synthesis, energy generation and respiration pathways. Interplay between these factors may play a key role in shaping the structure of microbial communities developed after oil spills at low depth and limit their bioremediation potential.

Up-scaling aquaculture wastewater treatment by microalgal bacterial flocs: from lab reactors to an outdoor raceway pond.

Sequencing batch reactors with microalgal bacterial flocs (MaB-floc SBRs) are a novel approach for photosynthetic aerated wastewater treatment based on bioflocculation. To assess their technical potential for aquaculture wastewater treatment in Northwest Europe, MaB-floc SBRs were up-scaled from indoor photobioreactors of 4 L over 40 and 400 L to a 12 m(3) outdoor raceway pond. Scale-up decreased the nutrient removal efficiencies with a factor 1-3 and the volumetric biomass productivities with a factor 10-13. Effluents met current discharge norms, except for nitrite and nitrate. Flue gas sparging was needed to decrease the effluent pH. Outdoor MaB-flocs showed enhanced settling properties and an increased ash and chlorophyll a content. Bioflocculation enabled successful harvesting by gravity settling and dewatering by filtering at 150-250 µm. Optimisation of nitrogen removal and biomass valorisation are future challenges towards industrial implementation of MaB-floc SBRs for aquaculture wastewater treatment.

Methyloparacoccus murrellii gen. nov., sp. nov., a methanotroph isolated from pond water.

Two novel methanotrophic strains, R-49797(T) and OS501, were isolated from pond water in South Africa and Japan, respectively. Strains R-49797(T) and OS501 shared 99.7% 16S rRNA gene sequence similarity. Cells were Gram-stain-negative, non-motile cocci with a diplococcoid tendency and contained type I methanotroph intracytoplasmic membranes. The pmoA gene encoding particulate methane monooxygenase was present. Soluble methane monoooxygenase (sMMO) activity, the mmoX gene encoding sMMO and the nifH gene encoding nitrogenase were not detected. Methane and methanol were utilized as sole carbon source. The strains grew optimally at 25-33 °C (range 20-37 °C) and at pH 6.3-6.8 (range 5.8-9.0). The strains did not support growth in media supplemented with 1% (w/v) NaCl. For both strains, the two major fatty acids were C(16 : 1)ω7c and C(16 : 0) and the DNA G+C content was 65.6 mol%. The isolates belong to the family Methylococcaceae of the class Gammaproteobacteria and cluster most closely among the genera Methylocaldum, Methylococcus and Methylogaea, with a 16S rRNA gene sequence similarity of 94.2% between strain R-49797(T) and its closest related type strain (Methylocaldum gracile VKM 14L(T)). Based on the low 16S rRNA gene sequence similarities with its nearest phylogenetic neighbouring genera, the formation of a separate lineage based on 16S rRNA and pmoA gene phylogenetic analysis, and the unique combination of phenotypic characteristics of the two isolated strains compared with the genera Methylocaldum, Methylococcus and Methylogaea, we propose to classify these strains as representing a novel species of a new genus, Methyloparacoccus murrellii gen. nov., sp. nov., within the family Methylococcaceae. The type strain of Methyloparacoccus murrellii is R-49797(T) ( = LMG 27482(T) = JCM 19379(T)).

Bioremediation of Southern Mediterranean oil polluted sites comes of age.

Mediterranean Sea is facing a very high risk of oil pollution due to the high number of oil extractive and refining sites along the basin coasts, and the intense maritime traffic of oil tankers. All the Mediterranean countries have adopted severe regulations for minimizing pollution events and bioremediation feasibility studies for the most urgent polluted sites are undergoing. However, the analysis of the scientific studies applying modern 'meta-omics' technologies that have been performed on marine oil pollution worldwide showed that the Southern Mediterranean side has been neglected by the international research. Most of the studies in the Mediterranean Sea have been done in polluted sites of the Northern side of the basin. Those of the Southern side are poorly studied, despite many of the Southern countries being major oil producers and exporters. The recently EU-funded research project ULIXES has as a major objective to increase the knowledge of the bioremediation potential of sites from the Southern Mediterranean countries. ULIXES is targeting four major polluted sites on the coastlines of Egypt, Jordan, Morocco and Tunisia, including seashore sands, lagoons, and oil refinery polluted sediments. The research is designed to unravel, categorize, catalogue, exploit and manage the diversity and ecology of microorganisms thriving in these polluted sites. Isolation of novel hydrocarbon degrading microbes and a series of state of the art 'meta-omics' technologies are the baseline tools for improving our knowledge on biodegradation capacities mediated by microbes under different environmental settings and for designing novel site-tailored bioremediation approaches. A network of twelve European and Southern Mediterranean partners is cooperating for plugging the existing gap of knowledge for the development of novel bioremediation processes targeting such poorly investigated polluted sites.

Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species.

The presence of persister cells has been proposed as a factor in biofilm resilience. In the present study we investigated whether persister cells are present in Burkholderia cepacia complex (Bcc) biofilms, what the molecular basis of antimicrobial tolerance in Bcc persisters is, and how persisters can be eradicated from Bcc biofilms. After treatment of Bcc biofilms with high concentrations of various antibiotics often a small subpopulation survived. To investigate the molecular mechanism of tolerance in this subpopulation, Burkholderia cenocepacia biofilms were treated with 1024 µg/ml of tobramycin. Using ROS-specific staining and flow cytometry, we showed that tobramycin increased ROS production in treated sessile cells. However, approximately 0.1% of all sessile cells survived the treatment. A transcriptome analysis showed that several genes from the tricarboxylic acid cycle and genes involved in the electron transport chain were downregulated. In contrast, genes from the glyoxylate shunt were upregulated. These data indicate that protection against ROS is important for the survival of persisters. To confirm this, we determined the number of persisters in biofilms formed by catalase mutants. The persister fraction in ΔkatA and ΔkatB biofilms was significantly reduced, confirming the role of ROS detoxification in persister survival. Pretreatment of B. cenocepacia biofilms with itaconate, an inhibitor of isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, reduced the persister fraction approx. 10-fold when the biofilms were subsequently treated with tobramycin. In conclusion, most Bcc biofilms contain a significant fraction of persisters that survive treatment with high doses of tobramycin. The surviving persister cells downregulate the TCA cycle to avoid production of ROS and at the same time activate an alternative pathway, the glyoxylate shunt. This pathway may present a novel target for combination therapy.

Decentralized two-stage sewage treatment by chemical-biological flocculation combined with microalgae biofilm for nutrient immobilization in a roof installed parallel plate reactor.

In this lab-scale study, domestic wastewater is subjected to a chemical biological adsorption (A-stage), followed by treatment in an innovative roof installed parallel plate microalgae biofilm reactor for nutrient immobilization (I-stage). The A-stage process was operated at a hydraulic retention time (HRT) of 1h and a solid retention time of 1day (FeSO(4) as flocculant). The I-stage, which consequently received the effluent of the A-stage process, was operated at an HRT of 1day and exposed to natural light. The overall system removed on average 74% of the total chemical oxygen demand, 82% of the total suspended solids, 67% of the total nitrogen and 96% of the total phosphorous in the wastewater. The design involves a relatively low capital and operating cost which is in the order of 0.5€/m(3) wastewater treated. These aspects suggest that the A/I process can be used as a decentralized domestic wastewater treatment system.