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In recent years, whole-cell biosensors (WCBs) have emerged as a potent approach for environmental monitoring and on-site analyte detection. These biosensors harness the biological apparatus of microorganisms to identify specific analytes, offering advantages in sensitivity, specificity, and real-time monitoring capabilities. A critical hurdle in biosensor development lies in ensuring the robust attachment of cells to surfaces, a crucial step for practical utility. In this study, we present a comprehensive approach to tackle this challenge via engineering Escherichia coli cells for immobilization on paper through the Curli biofilm pathway. Furthermore, incorporating a cellulose-binding peptide domain to the CsgA biofilm protein enhances cell adhesion to paper surfaces, consequently boosting biosensor efficacy. To demonstrate the versatility of this platform, we developed a WCB for copper, optimized to exhibit a discernible response, even with the naked eye. To confirm its suitability for practical field use, we characterized our copper sensor under various environmental conditions-temperature, salinity, and pH-to mimic real-world scenarios. The biosensor-equipped paper discs can be freeze-dried for deployment in on-site applications, providing a practical method for long-term storage without loss of sensitivity paper discs demonstrate sustained functionality and viability even after months of storage with 5 μM limit of detection for copper with visible-to-naked-eye signal levels. Biofilm-mediated surface attachment and analyte sensing can be independently engineered, allowing for flexible utilization of this platform as required. With the implementation of copper sensing as a proof-of-concept study, we underscore the potential of WCBs as a promising avenue for the on-site detection of a multitude of analytes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Urartu Ozgur Safak Seker reports financial support was provided by "The Scientific and Technological Research Council of Turkey". If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We reported a colorimetric paper-based device by integrating the modified acid RNA-cleaving DNAzymes (MaRCD-EC1) for highly sensitive (detection limit = 10 2 CFU mL -1 ), and rapid (within 30 min) detection of E. coli without amplification. This device exhibited a clinical sensitivity of 100% and a specificity of 100% in identifying E. coli -associated urinary tract infections (UTIs) using the clinical urine samples.

Many microbial phenotypes are differentially or exclusively expressed on agar surfaces, including biofilms, motility, and sociality. However, agar-based assays are limited by their low throughput, which increases costs, lab waste, space requirements, and the time required to conduct experiments. Here, we demonstrate the use of wax-printed microfluidic paper-based analytical devices (μPADs) to measure linear growth rate of microbes on an agar growth media as a means of circumventing the aforementioned limitations. The main production materials of the proposed μPAD design are a wax printer, filter paper, and empty pipet boxes. A single wax-printed μPAD allowing 8 independent, agar-grown colonies costs $0.07, compared to $0.20 and $9.37 for the same number of replicates on traditional microtiter/spectrophotometry and Petri dish assays, respectively. We optimized the μPAD design for channel width (3 mm), agar volume (780 μL/channel), and microbe inoculation method (razor-blade). Comparative analyses of the traditional and proposed μPAD methods for measuring growth rate of nonmotile (Saccharomyces cerevisiae) and motile (flagellated Escherichia coli) microorganisms suggested the μPAD assays conferred a comparable degree of accuracy and reliability to growth rate measurements as their traditional counterparts. We substantiated this claim with strong, positive correlations between the traditional and μPAD assay, a significant nonzero slope in the model relating the two assays, a nonsignificant difference between the relative standard errors of the two techniques, and an analysis of inter-device reliability. Therefore, μPAD designs merit consideration for the development of enhanced-throughput, low-cost microbial growth and motility assays., Competing Interests: The authors have declared that no competing interests exist.

Antimicrobial stewardship practices are critical in preventing the further erosion of treatment options for bacterial infections. Yet, at the same time, determination of an infection's antimicrobial susceptibility requires multiple rounds of culture and expensive lab automation systems. In this work, we report the use of paper-based surface enhanced Raman spectroscopy (SERS) sensors and portable instrumentation to phenotypically discriminate multi-drug resistance with fewer culture steps than conventional clinical microbiology. Specifically, we demonstrate the identification of resistance to varying generations of β-lactam antibiotics by detecting the activity of particular β-lactamase enzymes in a multiplexed assay. The method utilizes molecular reporters that consist of β-lactams with SERS barcodes. Hydrolysis of the β-lactam by β-lactamase enzymes in the sample expels the barcode; the released sulfur-containing barcode is then detected via SERS. Using this approach, we demonstrate the differentiation of E. coli strains with (1) extended spectrum β-lactamase (ESBL), (2) narrow-spectrum β-lactamase, and (3) no resistance, using only a single measurement on a single sample. In addition, we experimentally validate an approach to expand the library of reporters through the simple chemical synthesis of new barcoded β-lactams. Importantly, the reported method determines the susceptibility based on phenotypic β-lactamase activity, which is aligned with current microbiology lab standards. This new method will enable the precise selection of effective β-lactam antibiotics (as opposed to defaulting to drugs of last resort) faster than current methods while using simple steps and low-cost portable instrumentation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Nowadays, thanks to nanotechnological progress, which itself guides us more and more closely toward not only the efficient design of innovative nanomaterials or nanostructures, but to the improvement of their functionality, we benefit from an important asset in the battle against pathogenic illnesses. Herein, we report a versatile biocompatible plasmonic nanoplatform based on a Whatman paper incorporating positively-charged gold nanospherical particles via the immersion approach. The morphological characterization of the as-engineered-plasmonic paper was examined by SEM (scanning electron microscopy) and HRTEM (high-resolution transmission electron microscopy) investigations, while its surface chemical modification with a synthetic polypeptide, specifically RRWHRWWRR-NH2 (P2), was proved by monitoring the plasmonic response of loaded gold nanospheres and the emission signal of P2 via fluorescence spectroscopy. The as-functionalized plasmonic paper is non-cytotoxic towards BJ fibroblast human cells at bactericidal concentrations. Finally, the antimicrobial activity of the P2-functionalized plasmonic paper on both planktonic bacteria and biofilms was tested against two reference strains: Gram-positive Bacteria, i.e., Staphylococcus aureus and the Gram-negative Bacteria, i.e., Escherichia coli , determining microbial inhibition of up to 100% for planktonic bacteria. In line with the above presented nanoplatform's proper design, followed by their functionalization with active antimicrobial peptides, new roads can be open for determining antibiotic-free treatments against different relevant pathogens.

We report a microfluidic paper based analytical device implementing ion concentration polarization (ICP) for rapid pre-concentration of Escherichia coli in water. The fabricated device consists of a paper channel with a Nafion ® membrane and in-built micro wire electrodes to supply electric voltage to induce the ICP effect. E. coli cells were stained with SYTO 9 and fluorescence was used as a sensing method. The device achieved high concentration factor up to 2 × 10 5 within minutes. The effect of total ion concentration, on ICP and fluorescence intensity was studied. The reported device and method are suitable and effective for detection of E. coli during ballast water quality monitoring, coastal water quality monitoring where high salinity water is present., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The increasing prevalence of antibiotic-resistant bacteria needs rapid identification and efficient destruction routes. This study proposes testing paper derived from electrospun fibrous mats and aggregation-induced emission (AIE) probes for trace sensing and simultaneous destruction of antibiotic-resistant E. coli. Aptamers are conjugated on fibers for selective capture of E. coli, and the capture capability can be regenerated via rinsing with salt solution. Hydroxyl tetraphenylethene (TPE) is linked with two cephalosporin molecules to construct TPE-Cep probes, and the fluorescence emission is turned on specifically in the presence of β-lactamase, which is a critical marker for screening resistant bacteria. Fibrous mats are lit up only in the presence of antibiotic-resistant bacteria, and the fluorescence intensity changes could be statistically fitted into an equation for quantitative analysis. Fibrous strips display apparent color changes from blue to green for a visual readout of bacterial levels, and the limit of detection (LOD) is much lower than those of previous paper substrates. In addition, the TPE-Cep probes could produce reactive oxygen species (ROS) under room light illumination to kill the captured bacteria. Thus, the integration of aptamer-grafted electrospun fibers and functional AIE probes provides potential for selective capture, trace imaging and photodynamic destruction of antibiotic-resistant bacteria.

ATP-driven bioluminescence relying on the D-luciferin-luciferase reaction is widely employed for several biosensing applications where bacterial ATP detection allows to verify microbial contamination for hygiene monitoring in hospitals, food processing and in general for cell viability studies. Several ATP kit assays are already commercially available but an user-friendly ATP biosensor characterized by low-cost, portability, and adequate sensitivity would be highly valuable for rapid and facile on site screening. Thanks to an innovative freeze-drying procedure, we developed a user-friendly, ready-to-use and stable ATP sensing paper biosensor that can be combined with smartphone detection. The ATP sensing paper includes a lyophilized "nano-lantern" with reaction components being rapidly reconstituted by 10 μL sample addition, enabling detection of 10 -14  mol of ATP within 10 min. We analysed urinary microbial ATP as a biomarker of urinary tract infection (UTI), confirming the capability of the ATP sensing paper to detect the threshold for positivity corresponding to 10 5  colony-forming units of bacteria per mL of urine., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A large amount of wastewater containing various organic compounds is generated in the paper-making industry. Therefore, the re-utilization of wastewater from paper-making is of great importance. In this study, acetic acid and formic acid, the dominant inhibitors existing in the paper mill wastewater, were used to produce succinate by metabolically engineered Escherichia coli strains, HB04(pTrc99a-gltA) and HB04(pTrc99a-gltA, pBAD33-Trc-fdh). The distilled paper mill wastewater could be utilized and transformed directly to succinate efficiently. However, the utilization of acetate was inhibited dramatically using raw paper mill wastewater. After adsorption by activate carbon, the acetate consumption and succinate production were improved significantly. In the resting cells experiment with high cell density (50 OD 600 ), acetate was consumed completely and the titer of succinate reached 65.44 mM in 6 h. These results showed that the metabolically engineered E. coli strains had great potential to utilize acetic acid in the paper mill wastewater for succinate biosynthesis.

Laboratory support for low-resource regions is a rising global issue. As microbiological contamination is closely associated with other issues like food safety, water supply sustainability, and public health, bacterial assessments in this setting need to be improved. Herein, we demonstrate a paper-based diagnostic device for point-of-need testing, in which fecal-indicating Escherichia coli and highly pathogenic E. coli are detected by duplex coloration. This device was functionalized by mixing different chromogenic substrates that reflect each bacterial enzymatic phenotype. In the final part of the paper, we describe this microbiological diagnostic system tested with bacteria-contaminated food samples. The device sensitivity was shown to have greatly reduced the total analysis time (below to 4 h) when combined with an enrichment amplification procedure. Notably, this paper device successfully detected 10 cfu/mL of target bacteria in a contaminated milk sample. Our diagnostic system shows acceptable accuracy, short analysis time, and a user-friendly interface, thereby eliminating demands for high-end equipment and a highly trained staff. We expect that this diagnostic system will be a sustainable solution in supporting microbiological or clinical laboratories in low-income countries.

A novel microfluidic paper-based analytical device (μPAD) was developed with benzoquinone (BQ)-mediated E. coli respiration method to measure the biotoxicities of pollutants. Functional units including sample injection, fluid-cell separation, all-carbon electrode-enabled electrochemical detection, were integrated on a piece of chromatography paper. The three-electrode, working electrode, counter electrode and reference electrode, were simultaneously screen-printed on the μPAD with conductive carbon ink. The satisfying electrochemical performance of the paper-based carbon three-electrode was confirmed by cyclic voltammetry detecting K 3 [Fe(CN) 6 ]. The process of cell toxication was considered that toxicants inhibited cell respiration and diminished the electrons on E. coli respiratory chain. It was quantitatively reflected by measuring oxidation current of hydroquinone (HQ) as a reduced state of the redox mediator BQ after the incubation of cells with pollutants. The current detection time, BQ concentration and E. coli incubation time were carefully optimized to establish the systematic optimized operations of BQ-mediated E. coli respiration method. Using the fabricated μPAD the half inhibitory concentration (IC 50 ) were Cu 2+ solution 13.5 μg mL -1 , Cu 2+ -soil 21.4 mg kg -1 , penicillin sodium-soil 85.1 mg kg -1 , and IC 30 of Pb 2+ solution was 60.0 μg mL -1 . Detection of pesticide residues in vegetable juices were accomplished in a similar way. The proposed method is fascinating on three points; 1) The generality in the biotoxicity detection depends on toxicants inducing cellular respiratory inhibition; 2) The portability and affordability make it convenient for practical applications, because of replacing incubators and centrifuges; 3) There is potential applicability in less-developed areas due to its simple operation and low-cost., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Point-of-care (POC) detection and diagnostic platforms provide critical information about health and safety conditions in austere and resource-limited settings in which medical, military, and disaster relief operations are conducted. In this work, low-cost paper materials commonly used in POC devices are coated with liquid-infused polymer surfaces and folded to produce geometries that precisely localize complex liquid samples undergoing concentration by evaporation. Liquid-infused polymer surfaces were fabricated by infusing silicone-coated paper with a chemically compatible polydimethylsiloxane oil to create a liquid overlayer. Tests on these surfaces showed no remaining bacterial cells after exposure to a sliding droplet containing a concentrated solution of Escherichia coli or Staphylococcus aureus, while samples without a liquid layer showed adhesion of both microdroplets and individual bacterial cells. Folding of the paper substrates with liquid-infused polymer surfaces into several functional 3D geometries enabled a clean separation and simultaneous concentration of a liquid containing rhodamine dye into discrete, predefined locations. When used with bacteria, which are known for their ability to adhere to nearly any surface type, functional geometries with liquid-infused polymer surfaces concentrated the cells at levels significantly higher than geometries with dry control surfaces. These results show the potential of synergistically combining paper-based materials with liquid-infused polymer surfaces for the manipulation and handling of complex samples, which may help the future engineering of POC devices.

This paper demonstrates a simple-in-struct- ure, cost-effective, and environment-friendly Microfluidic Paper-based Analytical Device for Microbial Biofuel Cell ( μ PAD-MBFC). It consists of a microchannel with biofuel cell (Escherichia.Coli) and an oxidant (aerated tap water) flowing co-parallelly over Multiwalled Carbon Nanotube (MWCNT)-based Bucky Paper (BP) electrodes using a self-capillary and co-laminar flow mechanism. The electrochemical studies, such as open circuit potential (OCP) and polarization were evaluated using a potentiostat. Various volumetric bacterial studies were also carried out to find out the best suitable optimal bacterial volume. Subsequently, the morphological and detailed element composition study of electrode surface was performed using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) techniques. This well-designed portable μ PAD-MBFC yields a maximum power density of 4 [Formula: see text]/cm 2 ( [Formula: see text]/cm 2 ) at 0.405 V over [Formula: see text] of culture and leveraging to its long-lasting potential to operate miniaturized microelectronics sensors and portable devices.

A green and facile method is presented for in situ synthesis of a novel photoluminescence-quenching nanopaper with a highly-efficient quenching ability, rapid reaction time and long-term storage. The as-prepared nanopaper is further used to construct an aptasensor platform with high performance, rapidness and robustness.

Point-of-care testing (POCT) for uropathogen detection and chemical screening has great benefits for the diagnosis of urinary tract infections (UTIs). The goal of this study was to develop a portable and inexpensive paper-based analytical device (PAD) for cultivating bacteria in situ and rapidly testing for nitrite on the same device. The PAD was fabricated using a wax printing technique to create a pattern on Whatman No. 1 filter paper, which was then combined with a cotton sheet to support bacterial growth. Nitrite detection was based on the principle of the Griess reaction, and a linear detection range of 0-1.6 mg/dL (R 2  = 0.989) was obtained. Scanning electron microscopy (SEM) analysis demonstrated that the bacteria were able to grow and formed a cluster on the cellulose fibres within 2 hours. The enzyme β-glucuronidase, which is specifically produced by Escherichia coli, was able to convert the pre-immobilized 5-bromo-4-chloro-3-indolyl-β-D-glucuronide sodium salt (X-GlcA), a colourless substrate, generating a blue colour. Under optimum conditions, the proposed device allowed bacterial concentrations in the range of 10 4 -10 7 colony forming units (CFU)/mL to be quantified within 6 hours. Moreover, the use of this device enables the identification of E. coli pathogens with selectivity in real urine samples. In conclusion, the PAD developed in this study for UTI screening provides a rapid, cost-effective diagnostic method for use in remote areas.

Although significant technological advancements have been made in the development of analytical biosensor chips for detecting bacterial strains (E. coli, S. Mutans and B. Subtilis), critical requirements i.e. limit of detection (LOD), fast time of response, ultra-sensitivity with high reproducibility and good shelf-life with robust sensing capability have yet to be met within a single sensor chip. In order to achieve these criteria, we present an electrically-receptive thermally-responsive (ER-TR) sensor chip comprised of simple filter paper used as substrate coated with composite of poly(N-isopropylacrylamide) polymer (PNIPAm) - graphene nanoplatelet (GR) followed by evaporation of Au electrodes for capturing both Gram-positive (S. mutans and B. subtilis) and Gram-negative (E. coli) bacterial cells in real-time. Autoclave water, tap water, lake water and milk samples were tested with ER-TR chip with and without bacterial strains at varying concentration range 10 1 -10 5 cells/mL. The sensor was integrated with in-house built printed circuit board (PCB) to transmit/receive electrical signals. The interaction of E. coli, S. mutans and B. subtilis cells with fibers of PNIPAm-GR resulted in a change of electrical resistance and the readout was monitored wirelessly in real-time using MATLAB algorithm. Finally, prepared ER-TR chip exhibited the reproducibility of 85-97% with shelf-life of up to four weeks after testing with lake water sample., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ligno-cellulosic enzymes like cellulase and xylanase have potential for modification of paper pulp characteristics. A low molecular weight cellulase - xylanase complex (14KDa) was co-produced using Escherichia coli SD5. Co-existence of these enzymes was found to be advantageous in paper pulp modification and in deinking applications. The cellulase and xylanase exhibited specific activities of 51.95 and 24.64U/mg protein of respectively. Defibrillations, crack formation and changes in functional groups was evident from the SEM and FT-IR analysis of paper pulp following the enzyme treatment. The enzyme facilitated a better reduction of Kappa number and Hexenuronic acid (Hex A) compared to earlier studies. A Δ brightness of approximately 10% were achieved in case of both cellulase and xylanase for different treatment time. The tear strength of recycled paper was also found to increase after the enzymatic treatment. This is the first report on the application of a un-engineered E. coli isolate co-producing cellulase-xylanase for paper industrial application., (Copyright © 2017. Published by Elsevier B.V.)

Switching between metabolically active and dormant states provides bacteria with protection from environmental stresses and allows rapid growth under favorable conditions. This rapid growth can be detrimental to the environment, e.g., pathogens in recreational lakes, or to industrial processes, e.g., fermentation, making it useful to quickly determine when the ratio of dormant to metabolically active bacteria changes. While a rapid increase in metabolically active bacteria can cause complications, a high number of dormant bacteria can also be problematic, since they can be more virulent and antibiotic-resistant. To determine the metabolic state of Escherichia coli and Salmonella Typhimurium, we developed two paper-based colorimetric assays. The color changes were based on oxidoreductases reducing tetrazolium salts to formazans, and alkaline phosphatases cleaving phosphates from nitrophenyl phosphate salt. Specifically, we added iodophenyl-nitrophenyl-phenyl tetrazolium salt (INT) and methylphenazinium methyl sulfate to metabolically active bacteria on paper and INT and para-nitrophenyl phosphate salt to dormant bacteria on paper. The color changed in less than 60 min and was generally visible at 10 3  CFU and quantifiable at 10 6  CFU. The color changes occurred in both bacteria, since oxidoreductases and alkaline phosphatases are common bacterial enzymes. On one hand, this feature makes the assays suitable to a wide range of applications, on the other, it requires specific capture, if only one type of bacterium is of interest. We captured Salmonella or E. coli with immobilized P22 or T4 bacteriophages on the paper, before detecting them at levels of 10 2 or 10 4  CFU, respectively. Determining the ratio of the metabolic state of bacteria or a specific bacterium at low cost and in a short time, makes this methodology useful in environmental, industrial and health care settings.

We have developed a new litmus paper test (DipTest) for detecting Escherichia coli (E. coli) in water samples by performing enzymatic reactions directly on the porous paper substrate. The paper strip consists of a long narrow piece of cellulose blotting paper coated with chemoattractant (at bottom edge), wax hydrophobic barrier (at the top edge), and custom formulated chemical reagents (at reaction zone immediately below the wax hydrophobic barrier). When the paper strip is dipped in water, E. coli in the water sample is attracted toward the paper strip due to a chemotaxic mechanism followed by the ascent along the paper strip toward the reaction zone due to a capillary wicking mechanism, and finally the capillary motion is arrested at the top edge of the paper strip by the hydrophobic barrier. The E. coli concentrated at the reaction zone of the paper strip will react with custom formulated chemical reagents to produce a pinkish-red color. Such a color change on the paper strip when dipped into water samples indicates the presence of E. coli contamination in potable water. The performance of the DipTest device has been checked with different known concentrations of E. coli contaminated water samples using different dip and wait times. The DipTest device has also been tested with different interfering bacteria and chemical contaminants. It has been observed that the different interfering contaminants do not have any impact on the DipTest, and it can become a potential solution for screening water samples for E. coli contamination at the point of source.

In this study, we investigated the antibacterial activity of silver-coated gold nanoparticles (Au-Ag NPs) immobilized on cellulose paper. Ag NPs are known to have strong antibacterial properties, while Au NPs are biocompatible and relatively simple to prepare. We made the Au-Ag NPs using a facile process called Ag enhancement, in which Au NPs serve as the nuclei for precipitation of a Ag coating, the thickness of which can be easily controlled by varying the ratio of the reactants. After synthesis, electron microscopy showed that the Au-Ag NPs displayed a core-shell structure, and that they could be successfully immobilized onto a cellulose membrane by heat treatment. We then investigated the antibacterial properties of this NP-coated cellulose paper against E. coli JM109. The inhibition rate, growth curve, and AATCC 100 activity test showed that cellulose paper coated with 15 nm Au-Ag NPs possessed excellent antibacterial activity against E. coli JM109. These results suggest that Au-Ag NPs immobilized on cellulose paper could be a valuable antibacterial technology for applications such as food packaging, clothing, wound dressings, and other personal care products.

This work describes disposable plasma generators made from metallized paper. The fabricated plasma generators with layered and patterned sheets of paper provide a simple and flexible format for dielectric barrier discharge to create atmospheric plasma without an applied vacuum. The porosity of paper allows gas to permeate its bulk volume and fuel plasma, while plasma-induced forced convection cools the substrate. When electrically driven with oscillating peak-to-peak potentials of ±1 to ±10 kV, the paper-based devices produced both volume and surface plasmas capable of killing microbes. The plasma sanitizers deactivated greater than 99% of Saccharomyces cerevisiae and greater than 99.9% of Escherichia coli cells with 30 s of noncontact treatment. Characterization of plasma generated from the sanitizers revealed a detectable level of UV-C (1.9 nW⋅cm -2 ⋅nm -1 ), modest surface temperature (60 °C with 60 s of activation), and a high level of ozone (13 ppm with 60 s of activation). These results deliver insights into the mechanisms and suitability of paper-based substrates for active antimicrobial sanitization with scalable, flexible sheets. In addition, this work shows how paper-based generators are conformable to curved surfaces, appropriate for kirigami-like "stretchy" structures, compatible with user interfaces, and suitable for sanitization of microbes aerosolized onto a surface. In general, these disposable plasma generators represent progress toward biodegradable devices based on flexible renewable materials, which may impact the future design of protective garments, skin-like sensors for robots or prosthetics, and user interfaces in contaminated environments., Competing Interests: Conflict of interest statement: US Provisional Patent was filed (62/291,082) for “Low-Cost, Flexible, Paper-Based Plasma Sterilizer” on February 4, 2016. US Patent Application was filed (15/425,474) for “Flexible Plasma Applicators Based on Fibrous Layers” on February 6, 2017.

The development of transparency-based electrochemical and paper-based colorimetric analytic detection platforms is presented as complementary methods for food and waterborne bacteria detection from a single assay. Escherichia coli and Enterococcus species, both indicators of fecal contamination, were detected using substrates specific to enzymes produced by each species. β-galactosidase (β-gal) and β-glucuronidase (β-glucur) are both produced by E. coli, while β-glucosidase (β-gluco) is produced by Enterococcus spp. Substrates used produced either p-nitrophenol (PNP), o-nitrophenol (ONP), or p-aminophenol (PAP) as products. Electrochemical detection using stencil-printed carbon electrodes (SPCEs) was found to provide optimal performance on inexpensive and disposable transparency film platforms. Using SPCEs, detection limits for electrochemically active substrates, PNP, ONP, and PAP were determined to be 1.1, 2.8, and 0.5 μM, respectively. A colorimetric paper-based well plate system was developed from a simple cardboard box and smart phone for the detection of PNP and ONP. Colorimetric detection limits were determined to be 81 μM and 119 μM for ONP and PNP respectively. While colorimetric detection methods gave higher detection limits than electrochemical detection, both methods provided similar times to positive bacteria detection. Low concentrations (10 1 CFU/mL) of pathogenic and nonpathogenic E. coli isolates and (10 0 CFU/mL) E. faecalis and E. faecium strains were detected within 4 and 8 h of pre-enrichment. Alfalfa sprout and lagoon water samples served as model food and water samples, and while water samples did not test positive, sprout samples did test positive within 4 h of pre-enrichment. Positive detection of inoculated (2.3 × 10 2 and 3.1 × 10 1 CFU/mL or g of E. coli and E. faecium, respectively) sprout and water samples tested positive within 4 and 12 h of pre-enrichment, respectively.

This paper presents a pressed paper-based dipstick that enables detection of foodborne pathogens with multistep reactions by exploiting the delayed fluid flow and channel partition formation on nitrocellulose (NC) membrane. Fluid behaviors are easily modified by controlling the amount of pressure and the position of pressed region on the NC membrane. Detection region of the dipstick is optimized by controlling flow rate and delayed time based on Darcy's law. All the reagents required for assay are dried on the NC membrane and they are sequentially rehydrated at the prepartitioned regions when the device is dipped into sample solution. In this manner, multistep reactions can be facilitated by one-step dipping of the dipstick into the sample solution. As a proof of concept, we performed detection of two fatal foodborne pathogens (e.g., Escherichia coli O157:H7 and Salmonella typhimurium) with signal enhancement. In addition, we expanded the utilization of channel partitions by developing a pressed paper-based dipstick into dual detection format.

Water quality assessment requires a continuous and strict analysis of samples to guarantee compliance with established standards. Nowadays, the increasing number of pollutants and their synergistic effects lead to the development general toxicity bioassays capable to analyse water pollution as a whole. Current general toxicity methods, e.g. Microtox(®), rely on long operation protocols, the use of complex and expensive instrumentation and sample pre-treatment, which should be transported to the laboratory for analysis. These requirements delay sample analysis and hence, the response to avoid an environmental catastrophe. In an attempt to solve it, a fast (15 min) and low-cost toxicity bioassay based on the chromatic changes associated to bacterial ferricyanide reduction is here presented. E. coli cells (used as model bacteria) were stably trapped on low-cost paper matrices (cellulose-based paper discs, PDs) and remained viable for long times (1 month at -20 °C). Apart from bacterial carrier, paper matrices also acted as a fluidic element, allowing fluid management without the need of external pumps. Bioassay evaluation was performed using copper as model toxic agent. Chromatic changes associated to bacterial ferricyanide reduction were determined by three different transduction methods, i.e. (i) optical reflectometry (as reference method), (ii) image analysis and (iii) visual inspection. In all cases, bioassay results (in terms of half maximal effective concentrations, EC50) were in agreement with already reported data, confirming the good performance of the bioassay. The validation of the bioassay was performed by analysis of real samples from natural sources, which were analysed and compared with a reference method (i.e. Microtox). Obtained results showed agreement for about 70% of toxic samples and 80% of non-toxic samples, which may validate the use of this simple and quick protocol in the determination of general toxicity. The minimum instrumentation requirements and the simplicity of the bioassay open the possibility of in-situ water toxicity assessment with a fast and low-cost protocol., (Copyright © 2016 Elsevier B.V. All rights reserved.)

With advances in point-of-care testing (POCT), lateral flow assays (LFAs) have been explored for nucleic acid detection. However, biological samples generally contain complex compositions and low amounts of target nucleic acids, and currently require laborious off-chip nucleic acid extraction and amplification processes (e.g., tube-based extraction and polymerase chain reaction (PCR)) prior to detection. To the best of our knowledge, even though the integration of DNA extraction and amplification into a paper-based biosensor has been reported, a combination of LFA with the aforementioned steps for simple colorimetric readout has not yet been demonstrated. Here, we demonstrate for the first time an integrated paper-based biosensor incorporating nucleic acid extraction, amplification and visual detection or quantification using a smartphone. A handheld battery-powered heating device was specially developed for nucleic acid amplification in POC settings, which is coupled with this simple assay for rapid target detection. The biosensor can successfully detect Escherichia coli (as a model analyte) in spiked drinking water, milk, blood, and spinach with a detection limit of as low as 10-1000 CFU mL(-1), and Streptococcus pneumonia in clinical blood samples, highlighting its potential use in medical diagnostics, food safety analysis and environmental monitoring. As compared to the lengthy conventional assay, which requires more than 5 hours for the entire sample-to-answer process, it takes about 1 hour for our integrated biosensor. The integrated biosensor holds great potential for detection of various target analytes for wide applications in the near future.

Escherichia coli is a generic indicator of fecal contamination, and certain serotypes cause food- and water-borne illness such as O157:H7. In the clinic, detection of bacteriuria, which is often due to E. coli, is critical before certain surgical procedures or in cases of nosocomial infection to prevent further adverse events such as postoperative infection or sepsis. In low- and middle-income countries, where insufficient equipment and facilities preclude modern methods of detection, a simple, low-cost diagnostic device to detect E. coli in water and in the clinic will have significant impact. We have developed a simple paper-based colorimetric platform to detect E. coli contamination in 5h. On this platform, the mean color intensity for samples with 10(5)cells/mL is 0.118±0.002 (n=4), and 0.0145±0.003 (P<0.01⁎⁎) for uncontaminated samples. This technique is less time-consuming, easier to perform, and less expensive than conventional methods. Thus, paper-based ELISA is an innovative point-of-care diagnostic tool to rapidly detect E. coli, and possibly other pathogens when customized as appropriate, especially in areas that lack advanced clinical equipment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

As a cost-effective alternative to silver nanoparticles, we have investigated the use of copper nanoparticles in paper filters for point-of-use water purification. This work reports an environmentally benign method for the direct in situ preparation of copper nanoparticles (CuNPs) in paper by reducing sorbed copper ions with ascorbic acid. Copper nanoparticles were quickly formed in less than 10 min and were well distributed on the paper fiber surfaces. Paper sheets were characterized by x-ray diffraction, scanning electron microscopy, energy dispersive x-ray spectroscopy, and atomic absorption spectroscopy. Antibacterial activity of the CuNP sheets was assessed for by passing Escherichia coli bacteria suspensions through the papers. The effluent was analyzed for viable bacteria and copper release. The CuNP papers with higher copper content showed a high bacteria reduction of log 8.8 for E. coli. The paper sheets containing copper nanoparticles were effective in inactivating the test bacteria as they passed through the paper. The copper levels released in the effluent water were below the recommended limit for copper in drinking water (1 ppm)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (µPADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the µPAD. Alternatively, digital images of the reacted µPADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24 hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Bactericidal filter papers offer the simplicity of gravity filtration to simultaneously eradicate microbial contaminants and particulates. We previously detailed the development of biocidal block copolymer micelles that could be immobilized on a filter paper to actively eradicate bacteria. Despite the many advantages offered by this system, its widespread use is hindered by its unknown mechanism of action which can result in non-reproducible outcomes. In this work, we sought to investigate the mechanism by which a certain percentage of Escherichia coli cells survived when passing through the bactericidal filter paper. Through the process of elimination, the possibility that the bacterial survival probability was controlled by the initial bacterial load or the existence of resistant sub-populations of E. coli was dismissed. It was observed that increasing the thickness or the number of layers of the filter significantly decreased bacterial survival probability for the biocidal filter paper but did not affect the efficiency of the blank filter paper (no biocide). The survival probability of bacteria passing through the antibacterial filter paper appeared to depend strongly on the number of collision between each bacterium and the biocide-loaded micelles. It was thus hypothesized that during each collision a certain number of biocide molecules were directly transferred from the hydrophobic core of the micelle to the bacterial lipid bilayer membrane. Therefore, each bacterium must encounter a certain number of collisions to take up enough biocide to kill the cell and cells that do not undergo the threshold number of collisions are expected to survive., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Paper chips for immunoassay were patterned by screen printing of polydimethylsiloxane (PDMS) or wax pencil drawing. The methods for paper chip patterning are cheap, convenient, rapid and suitable for most laboratories. The whole time for patterning a paper chip is no more than 10 min. Visible immunoassay for the detection of bacteria (Escherichia coli ) has been realized using the paper chip, on which the antibody for capturing E. Coli was immobilized on the detection zones of the paper chip, while the detection antibody was labeled with gold nanoparticles (AuNPs) as a signal reporter. After an immunological reaction, the AuNPs bound on the paper chip can effectively catalyse the reduction of silver ions during the silver enhancing step, generating a visible result that can be read by naked eyes. The quantitative results can be acquired by scanning the silver stained paper chip with a commercial scanner/or digital camera. The density of E. coli in water samples can be measured after calibrating the gray value of silver stained spots with the logarithmic number of bacteria. The time and reagents consumed on the paper chip immunoassay is much smaller than those of conventional ELISA, while the sensitivity of the paper chip immunoassay is comparable to conventional ELISA. The technology proposed in this work displays a great potential in the in-situ analysis when daily monitoring of water quality are required., (© 2013 Published by Elsevier B.V.)

To detect antibiotic-resistant bacteria in areas remote from microbiology laboratories, we designed portable culture devices performing an analogue of the Kirby-Bauer disk diffusion test inside patterned papers embedded in tape. We quantified the antibiotic susceptibility of several strains of Escherichia coli and Salmonella typhimurium by measuring blue-colored zones of inhibited growth.

An attempt has been made for enhancing the thermostability of xylanase (Mxyl) retrieved from a compost-soil-based metagenomic library. The analysis of the structure of xylanase by molecular dynamics simulation revealed more structural fluctuations in β-sheets. When the surface of β-sheets was enriched with arginine residues by substituting serine/threonine by site-directed mutagenesis, the enzyme with four arginine substitutions (MxylM4) exhibited enhanced thermostability at 80 °C. The T 1/2 of MxylM4 at 80 °C, in the presence of birchwood xylan, increased from 130 to 150 min at 80 °C without any alteration in optimum pH and temperature and molecular mass. Improvement in thermostability of MxylM4 was corroborated by increase in T m by 6 °C over that of Mxyl. The K m of MxylM4, however, increased from 8.01 ± 0.56 of Mxyl to 12.5 ± 0.32 mg ml(-1), suggesting a decrease in the affinity as well as specific enzyme activity. The Mxyl as well as MxylM4 liberated chromophores and lignin-derived compounds from kraft pulp, indicating their applicability in pulp bleaching.

We previously produced a bactericidal filter paper loaded with PAA47-b-PS214 block copolymer micelles containing the biocide triclosan (TCN), using cationic polyacryamide (cPAM) as a binder. However, we encountered a very slow filtration, resulting in long bacteria deactivation times. Slow drainage occurred only when the filter paper was left to dry. It appears that the filter paper with cPAM and micelles develops hydrophobic properties responsible for this very slow filtration. Three approaches were taken to accelerate the very slow drainage all based on modification of binder-micelle interactions: (i) keeping the micelles wet, (ii) modification of the corona, and (iii) replacing cPAM with smaller and more highly charged cationic poly(isopropanol dimethylammonium) chloride (PIDMAC). In all cases, the drainage time of bactericidal filter paper became close to that of untreated filter paper, without decreasing its efficiency. Moreover, replacing cPAM with PIDMAC led to a much more efficient bactericidal filter paper that reduced bacteria viability by more than 6 orders of magnitude. In addition to resolving the hydrophobic drainage hurdle, the three solutions also offer a better understanding of the interaction between cPAM and micelles in the filter paper.

To the authors' knowledge, the role of restaurant menus as a vehicle for pathogens has not been explored. Menus, however, can pose as a vector for bacterial contamination and transfer. Sampling menus from two restaurants in the Houston, Texas, area showed the presence of up to 100 CFU/cm2 aerobic bacteria. Follow-up studies designed to investigate the ability of Salmonella and E. coli to persist on paper and laminated menus at various time points (0, 6, 24, 48, and 72 hours) demonstrated that bacteria persist more efficiently on laminated menus as compared to paper menus. Transfer studies performed to quantitatively determine the ability of bacteria to transfer from menus to fingertips and from fingertips to clean menus showed that bacteria can be transferred for up to 24 hours. The study described here showed that restaurant menus may serve as vehicles for pathogens and hence present a public health issue within the retail food environment.

Rapid, sensitive, on-site detection of bacteria without a need for sophisticated equipment or skilled personnel is extremely important in clinical settings and rapid response scenarios, as well as in resource-limited settings. Here, we report a novel approach for selective and ultra-sensitive multiplexed detection of Escherichia coli (non-pathogenic or pathogenic) using a lab-on-paper test strip (bioactive paper) based on intracellular enzyme (β-galactosidase (B-GAL) or β-glucuronidase (GUS)) activity. The test strip is composed of a paper support (0.5 × 8 cm), onto which either 5-bromo-4-chloro-3-indolyl-β-D: -glucuronide sodium salt (XG), chlorophenol red β-galactopyranoside (CPRG) or both and FeCl(3) were entrapped using sol-gel-derived silica inks in different zones via an ink-jet printing technique. The sample was lysed and assayed via lateral flow through the FeCl(3) zone to the substrate area to initiate rapid enzyme hydrolysis of the substrate, causing a change from colorless-to-blue (XG hydrolyzed by GUS, indication of nonpathogenic E. coli) and/or yellow to red-magenta (CPRG hydrolyzed by B-GAL, indication of total coliforms). Using immunomagnetic nanoparticles for selective preconcentration, the limit of detection was ~5 colony-forming units (cfu) per milliliter for E. coli O157:H7 and ~20 cfu/mL for E. coli BL21, within 30 min without cell culturing. Thus, these paper test strips could be suitable for detection of viable total coliforms and pathogens in bathing water samples. Moreover, inclusion of a culturing step allows detection of less than 1 cfu in 100 mL within 8 h, making the paper tests strips relevant for detection of multiple pathogens and total coliform bacteria in beverage and food samples.

Background: Paper is omnipresent on hospital units, but few studies have examined the possible role of paper in the spread of nosocomial pathogens., Objective: To determine by laboratory investigation how long bacterial pathogens can survive on office paper and whether bacteria can be transferred from hands to paper and back to hands in a "worst-case scenario.", Methods: Samples of four bacterial pathogens (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus hirae) were prepared according to standard laboratory procedures. Sterile swatches of office paper were inoculated with the pathogens and bacterial survival was tested over seven days. To test the transmission of bacteria from one person's hands to paper and back to another person's hands, the fingertips of volunteers were inoculated with a nonpathogenic strain of E. coli; these volunteers then pressed the inoculum onto sterile paper swatches. Another group of volunteers whose hands had been moistened pressed their fingertips onto the contaminated paper swatches. Bacteria transferred to the moistened fingertips were cultivated according to standard laboratory procedures., Results: The four tested organisms showed differences in length of survival depending on environmental room conditions, but were stable on paper for up to 72 hours and still cultivable after seven days. Test organisms were transferred to paper, survived on it, and were retransferred back to hands., Conclusion: Paper can serve as a vehicle for cross-contamination of bacterial pathogens in medical settings if current recommendations on hand hygiene aren't meticulously followed.

Biosolids produced from pulp and paper mill wastewater treatment have excellent properties as soil conditioners, but often contain high levels of Escherichia coli. E. coli are commonly used as indicators of fecal contamination and health hazard; therefore, their presence in biosolids causes concern and has lead to restrictions in land-spreading. The objectives of this study were to determine the following: (1) if E. coli from the biosolids of a wastewater-free pulp and paper mill were enteric pathogens, and (2) if other waterborne microbial pathogens were present. E. coli were screened for heat-labile and heat-stable enterotoxin and verocytotoxin virulence genes using a polymerase chain reaction. Ten isolates were also screened for invasion-associated locus and invasion plasmid antigen H genes. None of the 120 isolates carried these genes. Tests for seven other microbial pathogens were negative. Effluents and biosolids from this mill do not contain common microbial pathogens and are unlikely to pose a health hazard.

To provide the first step towards a microbial ecology-based understanding of irreversible membrane biofouling, four laboratory-scale membrane bioreactors (MBRs) were operated to investigate the identity of bacterial populations highly correlated with irreversible membrane biofouling. The conventional MBR was divided into two separate experimental units. Unit one consisted of four suspended-growth, activated sludge, sequencing batch bioreactors treating a synthetic paper mill wastewater. Unit two consisted of a microfiltration membrane cell. Amplified ribosomal deoxyribonucleic acid restriction analysis (ARDRA) was used to compare the predominant bacterial populations in samples of mixed liquor and irreversibly bound to the membrane surface. The results of ARDRA showed a significant difference between the planktonic and sessile bacterial communities suggesting that irreversible biofouling of microfiltration membranes may be more highly correlated to specific bacterial populations rather than the total, bulk concentration of biomass. A custom-built mini-flow cell and light microscopy were used to visualise the early formation of biofilms by two pure cultures (Escherichia coli and Acinetobacter calcoaceticus) on membrane surfaces. The results confirmed that A. calcoaceticus was able to enhance the initiation of biofilm formation on microfiltration membranes.

Paper sludges are solid wastes material generated from the paper production, which have been characterized for their chemical contents. Some are rich in wood fiber and are a good carbon source, for example the primary and de-inking paper sludges. Others are made rich in nitrogen and phosphorus by pressing the activated sludge, resulting from the biological water treatments, with the primary sludge, yielding the combined paper sludge. Still, in the absence of sanitary effluents very few studies have addressed the characterization of their coliform microflora. Therefore, this study investigated the thermotolerant coliform population of one paper mill effluent and two paper mill sludges and wood chips screening rejects using chromogenic media. For the first series of analyses, the medium used was Colilert broth and positive tubes were selected to isolate bacteria in pure culture on MacConkey agar. In a second series of analyses, double selective media, based on ss-galactosidase and ss-glucuronidase activities, were used to isolate bacteria. First, the presence of thermotolerant coliforms was detected in low numbers in most water effluents, but showed that the entrance of the thermotolerant coliforms was early in the industrial process. Also, large numbers of thermotolerant coliforms, i.e., 7,000,000 MPN/g sludge (dry weight; d.w.), were found in combined sludges. From this first series of isolations, bacteria were purified on MacConkey medium and identified as Citrobacter freundii, Enterobacter sp, E. sakazakii, E. cloacae, Escherichia coli, K. pneumoniae, K. pneumoniae subsp. rhinoscleromatis, K. pneumoniae subsp. ozaenae, K. pneumoniae subsp. pneumoniae, Pantoea sp, Raoultella terrigena, R. planticola. Second, the presence of thermotolerant coliforms was measured at more than 3,700-6,000 MPN/g (d.w) sludge, whereas E. coli was detected from 730 to more than 3,300 MPN/g (d.w.) sludge. The presence of thermotolerant coliform bacteria and E. coli was sometimes detected from wood chips screening rejects in large quantities. Also, indigenous E. coli were able to multiply into the combined sludge, and inoculated E. coli isolates were often able to multiply in wood chips and combined sludge media. In this second series of isolations, API20E and Biolog identified most isolates as E. coli, but others remained unidentified. The sequences of the 16S rDNA confirmed that most isolates were likely E. coli, few Burkholderia spp, but 10% of the isolates remained unidentified. This study points out that the coliform bacteria are introduced by the wood chips in the water effluents, where they can survive throught the primary clarifier and regrow in combined sludges.

In order to determine desiccation tolerances of bacterial strains, the survival of 58 diarrheagenic strains (18 salmonellae, 35 Shiga toxin-producing Escherichia coli [STEC], and 5 shigellae) and of 15 nonpathogenic E. coli strains was determined after drying at 35 degrees C for 24 h in paper disks. At an inoculum level of 10(7) CFU/disk, most of the salmonellae (14/18) and the STEC strains (31/35) survived with a population of 10(3) to 10(4) CFU/disk, whereas all of the shigellae (5/5) and the majority of the nonpathogenic E. coli strains (9/15) did not survive (the population was decreased to less than the detection limit of 10(2) CFU/disk). After 22 to 24 months of subsequent storage at 4 degrees C, all of the selected salmonellae (4/4) and most of the selected STEC strains (12/15) survived, keeping the original populations (10(3) to 10(4) CFU/disk). In contrast to the case for storage at 4 degrees C, all of 15 selected strains (5 strains each of Salmonella spp., STEC O157, and STEC O26) died after 35 to 70 days of storage at 25 degrees C and 35 degrees C. The survival rates of all of these 15 strains in paper disks after the 24 h of drying were substantially increased (10 to 79 times) by the presence of sucrose (12% to 36%). All of these 15 desiccated strains in paper disks survived after exposure to 70 degrees C for 5 h. The populations of these 15 strains inoculated in dried foods containing sucrose and/or fat (e.g., chocolate) were 100 times higher than those in the dried paper disks after drying for 24 h at 25 degrees C.

The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known beta-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca2+ and showed its maximum at 53 degrees C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect.

We report the results of an interdisciplinary collaboration formed to assess the sterilizing capabilities of the One Atmosphere Uniform Glow Discharge Plasma (OAUGDP). This newly-invented source of glow discharge plasma (the fourth state of matter) is capable of operating at atmospheric pressure in air and other gases, and of providing antimicrobial active species to surfaces and workpieces at room temperature as judged by viable plate counts. OAUGDP exposures have reduced log numbers of bacteria, Staphylococcus aureus and Escherichia coli, and endospores from Bacillus stearothermophilus and Bacillus subtilis on seeded solid surfaces, fabrics, filter paper, and powdered culture media at room temperature. Initial experimental data showed a two-log10 CFU reduction of bacteria when 2 x 10(2) cells were seeded on filter paper. Results showed > or = 3 log10 CFU reduction when polypropylene samples seeded with E. coli (5 x 10(4)) were exposed, while a 30 s exposure time was required for similar killing with S. aureus-seeded polypropylene samples. The exposure times required to effect > or = 6 log10 CFU reduction of E. coli and S. aureus on polypropylene samples were no longer than 30 s. Experiments with seeded samples in sealed commercial sterilization bags showed little or no differences in exposure times compared to unwrapped samples. Plasma exposure times of less than 5 min generated > or = 5 log10 CFU reduction of commercially prepared Bacillus subtilis spores (1 x 10(5)); 7 min OAUGDP exposures were required to generate a > or = 3 log10 CFU reduction for Bacillus stearothermophilus spores. For all microorganisms tested, a biphasic curve was generated when the number of survivors vs time was plotted in dose-response cures. Several proposed mechanisms of killing at room temperature by the OAUGDP are discussed.

Escherichia coli KO11 was previously constructed to produce ethanol from acid hydrolysates of hemicellulose (pentoses and hexoses) by the chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB). Klebsiella oxytoca P2 was constructed in an analogous fashion for the simultaneous saccharification and fermentation of cellulose and contains PTS enzymes for cellobiose. In this study, KO11 was further engineered for the fermentation of cellulose by adding the K. oxytoca casAB genes encoding Enzyme IIcellobiose and phospho-beta-glucosidase. Although the two K. oxytoca genes were well expressed in cloning hosts such as DH5 alpha, both were expressed poorly in E. coli KO11, a derivative of E. coli B. Spontaneous mutants which exhibited more than 15-fold-higher specific activities for cellobiose metabolism were isolated. The mutations of these mutants resided in the plasmid rather than the host. Three mutants were characterized by sequence analysis. All contained similar internal deletions which eliminated the casAB promoter and operator regions and placed the lacZ Shine-Dalgarno region immediately upstream from the casA Shine-Dalgarno region. KO11 harboring mutant plasmids (pLOI1908, pLOI1909, or pLOI1910) rapidly fermented cellobiose to ethanol, and the yield was more than 90% of the theoretical yield. Two of these strains were used with commercial cellulase to ferment mixed-waste office paper to ethanol.

This article presents the advanced technology that has been developed by BioEnergy International of Gainesville, Florida, utilizing novel recombinant strains of bacteria developed by Lonnie Ingram of the University of Florida. The first commercial applications of these unique fermenting organisms convert 5-carbon sugars, as well as 6-carbon sugars, and oligomers of cellulose (e.g., cellobiose and cellotriose) directly to ethanol. The proposed systems that will be utilized for conversion of agricultural wastes, mixed waste papers, and pulp and paper mill waste in forthcoming commercial installations are now under design. This involves the extensive experience of Raphael Katzen Associates International, Inc. in acid hydrolysis, enzyme production, enzymatic hydrolysis, large-scale fermentation engineering, and distillation/dehydration. Specific examples of this advanced technology will be presented in different applications, namely: 1. Conversion of the hemicellulose content of sugar cane bagasse to 5-carbon sugars by mild-acid prehydrolysis, followed by fermentation of the 5-carbon sugar extract with recombinant Escherichia coli in a commercial installation soon to be under construction in Brazil. This unique process utilizes the surplus hemicellulose fraction of bagasse not required for steam and power generation to produce ethanol, additional to that from the original can juice, which has been converted by conventional sucrose fermentation to ethanol. The process also recovers and converts to ethanol the majority of sucrose normally lost with the bagasse fibers. Resultant beer is enriched in an innovative process to eliminate the need for incremental rectification capacity. 2. Application of this technology to mixed waste paper in Florida, with a moderate loading of newsprint (85% mechanical wood fiber), will involve a mild-acid prehydrolysis, the partial extraction of the 5-carbon sugars produced from hemicellulose as a feedstock for propagation of the recombinant Klebsiella oxytoca bacterium. Included is a facility providing for in-house production of cellulase enzyme, as an active whole broth for direct use in simultaneous saccharification and fermentation (SSF) of the remaining cellulose and residual 5-carbon sugars to ethanol. This is followed by distillation and dehydration in the advanced commercially available low-energy recovery system. 3. Another potential application of this unique technology involves utilization of a variety of wastes from several pulp and paper mills in close proximity, permitting collection of these wastes at low cost and reducing the considerable cost encountered in disposing of such low-energy wet waste.(ABSTRACT TRUNCATED AT 400 WORDS)

The coliform flora of a pulp and cardboard mill that uses birch as the raw material and ammonium sulphate as the process chemical was studied. Escherichia coli was observed to multiply in the mill. It persisted as the dominant thermotolerant coliform in the effluent. Klebsiellae were encountered among total coliforms only. The E. coli strains isolated had the biochemical characteristics and maximum growth temperatures typical to the species. However, serotyping and hemolysin test differentiated these strains from pathogenic and fecal E. coli.

A rapid assay for tRNA sulfurtransferase from Escherichia coli was developed, reducing the time needed to determine enzyme activity from 11 to 2 h. The reaction measured is the transfer of sulfur from [35S]cysteine to acceptor sites in a thionucleotide-deficient tRNA substrate. Processing is done by binding the product, [35S]-tRNA, to DEAE-cellulose filter disks. The disks are then treated to remove unreacted [35S]cysteine, cysteine-protein adducts and [35S]cysteinyl-tRNA. The DE81 disk assay and the 11-h standard assay are shown to give identical values over a wide range of incubation times and enzyme levels. Incorporation was greater when thionucleotide-deficient tRNA was used as substrate, as compared to fully modified tRNA. [35S]-tRNA was found to be the major reaction product, although some [35S]cysteine was also bound to the filters. The major thionucleoside labeled in nucleoside digests was 4-thiouridine, as determined by Bio-Gel P2 chromatography. We also observed other labeled peaks by this method, in amounts too small for positive identification. This rapid assay should be useful in the purification and study of this uncharacterized class of tRNA modification enzymes.

Milk specimens, 75 from cows immunized against cholera toxin and 35 from a human population in which enterotoxigenic Escherichia coli and rotaviral infections are endemic, were collected as paired filter paper and frozen whole milk samples. Each pair was tested for antibody activity against heat-labile E. coli and Vibrio cholerae enterotoxins. Additionally, 12 of the 35 paired human milk samples stored as frozen whole milk and dried on filter paper were tested for anti-rotavirus immunoglobulin A. Anti-enterotoxin and anti-rotavirus immunoglobulin A titers in milk dried on filter paper compared favorably with those of their frozen whole milk pairs. Filter paper samples offered considerable advantages for field collection, transportation, and storage over frozen liquid samples.