Removal of microbial multi-species biofilms from the paper industry by enzymatic treatments

This study aimed to characterize biofilms from the paper industry and evaluate the effectiveness of enzymatic treatments in reducing them. The extracellular polymeric substances (EPS) extracted from six industrial biofilms were studied. EPS were mainly proteins, the protein to polysaccharide ratio ranging from 1.3 to 8.6 depending on where the sampling point was situated in the paper making process. Eight hydrolytic enzymes were screened on a 24-h multi-species biofilm. The enzymes were tested at various concentrations and contact durations. Glycosidases and lipases were inefficient or only slightly efficient for biofilm reduction, while proteases were more efficient: after treatment for 24 h with pepsin, Alcalase® or Savinase®, the removal exceeded 80%. Savinase® appeared to be the most adequate for industrial conditions and was tested on an industrial biofilm sample. This enzyme led to a significant release of proteins from the EPS matrix, indicating its potential efficiency on an industrial scale.     

Using enzymes to remove biofilms of bacterial isolates sampled in the food-industry

The aim of this study was to analyze the cleaning efficiency of polysaccharidases and proteolytic enzymes against biofilms of bacterial species found in food industry processing lines and to study enzyme effects on the composition of extracellular polymeric substances (EPS) and biofilm removal in a Clean-in-Place (CIP) procedure. The screening of 7 proteases and polysaccharidases for removal of biofilms of 16 bacterial species was first evaluated using a microtiter plate assay. The alkaline pH buffer removed more biofilm biomass as well as affecting a larger range of bacterial species. The two serine proteases and α-amylase were the most efficient enzymes. Proteolytic enzymes promoted biofilm removal of a larger range of bacterial species than polysaccharidases. Using three isolates derived from two bacterial species widely found in food processing lines (Pseudomonas fluorescens and the Bacillus cereus group), biofilms were developed on stainless steel slides and enzymatic solutions were used to remove the biofilms using CIP procedure. Serine proteases were more efficient in removing cells of Bacillus biofilms than polysaccharidases. However, polysaccharidases were more efficient in removing P. fluorescens biofilms than serine proteases. Solubilization of enzymes with a buffer containing surfactants, and dispersing and chelating agents enhanced the efficiency of polysaccharidases and proteases respectively in removing biofilms of Bacillus and P. fluorescens. A combination of enzymes targeting several components of EPS, surfactants, dispersing and chelating agents would be an efficient alternative to chemical cleaning agents.     

Characterisation of the Physical Composition and Microbial Community Structure of Biofilms within a Model Full-Scale Drinking Water Distribution System

Within drinking water distribution systems (DWDS), microorganisms form multi-species biofilms on internal pipe surfaces. A matrix of extracellular polymeric substances (EPS) is produced by the attached community and provides structure and stability for the biofilm. If the EPS adhesive strength deteriorates or is overcome by external shear forces, biofilm is mobilised into the water potentially leading to degradation of water quality. However, little is known about the EPS within DWDS biofilms or how this is influenced by community composition or environmental parameters, because of the complications in obtaining biofilm samples and the difficulties in analysing EPS. Additionally, although biofilms may contain various microbial groups, research commonly focuses solely upon bacteria. This research applies an EPS analysis method based upon fluorescent confocal laser scanning microscopy (CLSM) in combination with digital image analysis (DIA), to concurrently characterize cells and EPS (carbohydrates and proteins) within drinking water biofilms from a full-scale DWDS experimental pipe loop facility with representative hydraulic conditions. Application of the EPS analysis method, alongside DNA fingerprinting of bacterial, archaeal and fungal communities, was demonstrated for biofilms sampled from different positions around the pipeline, after 28 days growth within the DWDS experimental facility. The volume of EPS was 4.9 times greater than that of the cells within biofilms, with carbohydrates present as the dominant component. Additionally, the greatest proportion of EPS was located above that of the cells. Fungi and archaea were established as important components of the biofilm community, although bacteria were more diverse. Moreover, biofilms from different positions were similar with respect to community structure and the quantity, composition and three-dimensional distribution of cells and EPS, indicating that active colonisation of the pipe wall is an important driver in material accumulation within the DWDS.     

Identification of biofilm matrix-associated proteins from an acid mine drainage microbial community

In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by ～2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as β-N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.     

Model system for growing and quantifying Streptococcus pneumoniae biofilms in situ and in real time

Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation. A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S. pneumoniae in situ over time. A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer. Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods. ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS). Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides. Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h. The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides. The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 10(5) cells per cm(2), while viable counts decreased as the biofilm aged. This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S. pneumoniae over time by using multiple, corroborative techniques. This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance.     

Proteomics for the analysis of the Candida albicans biofilm lifestyle

Candida albicans is an opportunistic pathogenic fungus capable of causing infections in immunocompromised patients. Candidiasis is often associated with the formation of biofilms on the surface of inert or biological materials. Biofilms are structured microbial communities attached to a surface and encased within a matrix of exopolymeric substance (EPS). At present, very little is known about the changes in protein profiles that occur during the transition from the planktonic to the biofilm mode of growth. Here, we report the use of proteomics for the comparative analysis of subcellular fractions obtained from C. albicans biofilm and planktonic cultures, including cell surface-associated proteins and secreted components present in liquid culture supernatants (for planktonic cultures) and EPS (for biofilms). The analysis revealed a high degree of similarity between the protein profiles associated with the planktonic and biofilm extracts, and led to the identification of several differentially expressed protein spots. Among the differentially expressed proteins, there was a preponderance of metabolic enzymes that have been described as cell surface proteins and immunodominant antigens. Proteins found in the biofilm matrix included a few predicted to form part of the secretome, and also many secretion-signal-less proteins. These observations contribute to our understanding of the C. albicans biofilm lifestyle.     

Distribution and composition of extracellular polymeric substances in membrane-aerated biofilm

Extracellular polymeric substances (EPS) are one of the main components of the biofilm and perform important functions in the biofilm system. In this study, two membrane-aerated biofilms (MABs) were developed for the thin and thick biofilms under different surface loading rates (SLRs). Supplies of oxygen and substrates in the MAB were from two opposite directions. This counter diffusion of nutrients resulted in a different growth environment, in contrast to conventional biofilms receiving both oxygen and substrates from the same side. The compositions, distributions and physicochemical properties (solubility and bindability) of EPS in the MABs of different thicknesses under different SLRs were studied. The effect of dissolved oxygen (DO) concentration within the MAB on EPS properties and distribution was investigated. Experimental results showed the different biofilm thicknesses produced substantially different profiles of EPS composition and distribution. Soluble proteins were more dominant than soluble polysaccharides in the inner aerobic layer of the biofilms; in contrast, bound proteins were greater than bound polysaccharides in the outer anoxic or anaerobic layer of the biofilms. The biofilm-EPS matrix consisted mainly of bound EPS. Bound EPS exhibited a hump-shaped profile with the highest content occurring in an intermediate region in the thin MAB and relatively more uniformly in the one half of the biofilm close to the membrane side and then declined towards the biofilm-liquid interface in the thick MAB. The profiles of soluble EPS presented a similar declining trend from the membrane towards the outer region in both thin and thick MABs. The study suggested that not only EPS composition but also EPS distribution and properties (solubility and bindability) played a crucial role in controlling the cohesiveness and maintaining the structural stability and stratification of the MABs.     

The role of exopolysaccharides in dual species biofilm development

A plasmid encoding the green fluorescent protein (GFP) of Aequorea victoria was transformed into a biofilm-forming strain of Enterobacter agglomerans originally isolated from an industrial environment. The transformed strain, EntGFP, could then be identified in dual species biofilms by direct visualization, plate counts and quantitiative fluorescence measurements. A variety of cell constituents and products may be involved in the adhesion and accumulation process and exopolysaccharides (EPS) represent one of these factors. The involvement of EPS in the initial adhesion events and the role in dual species biofilm development was investigated. Cells of EntGFP and Klebsiella pneumoniae Gl interact forming biofilms more successfully in a mixture than in isolation. The co-resistance results in enhanced biofilm formation and increased resistance to disinfection. Microscopic examination showed that the two species were often closely juxtaposed in microcolonies, suggesting the interactions involve surface-associated macromolecules. Fluorescence was used to measure the adhesion of EntGFP cells to Kleb, pneumoniae Gl (Gl) EPS. The results showed EntGFP adhered better to Gl EPS that Ent EPS. Polysaccharde depolymerases isolated from a bacteriophage for Ent. agglomerans were used to degrade Ent EPS specifically. Following polysaccharase treatment, the adhaesion of EntGFP to Gl cells was reduced. This suggests both types of EPS mediate adhesion. The two types of EPS were dissolved in dimethylsulphoxide and when mixed, their viscosity increased, reaching a maximum after ～+40 min. This may partially explain the increased protection of dual species biofilms from disinfectants. The depolymerases were used to treat dual species biofilms and this resulted in the effective removal of both species from the surface. This may suggest Ent contributes more EPS to the biofilm matrix. The EPS play an important role in EntGFP and Gl dual species biofilm formation both as adhesins and as the EPS interact, changing their physical properties.     

Persister cells in a biofilm treated with a biocide

This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC = 0.55 ± 0.20 mM for planktonic cells; MBC = 1.7 ± 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA, and the number of viable and culturable cells increased over time. The MBC of the recovered biofilm cells after a second exposure to OPA was 0.58 ± 0.40 mM, a concentration similar to the MBC of planktonic cells. This study demonstrates that persister cells may survive in biocide-treated biofilms, even in the absence of EPS.     

Model System for Growing and Quantifying Streptococcus pneumoniae Biofilms In Situ and in Real Time

Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation. A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S. pneumoniae in situ over time. A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer. Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods. ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS). Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides. Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h. The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides. The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 10⁵ cells per cm², while viable counts decreased as the biofilm aged. This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S. pneumoniae over time by using multiple, corroborative techniques. This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance.     

Mechanisms of Bacillus cereus biofilm formation: an investigation of the physicochemical characteristics of cell surfaces and extracellular proteins

Microbial biofilms contribute to biofouling in a wide range of processes from medical implants to processed food. The extracellular polymeric substances (EPS) are implicated in imparting biofilms with structural stability and resistance to cleaning products. Still, very little is known about the structural role of the EPS in Gram-positive systems. Here, we have compared the cell surface and EPS of surface-attached (biofilm) and free-floating (planktonic) cells of Bacillus cereus, an organism routinely isolated from within biofilms on different surfaces. Our results indicate that the surface properties of cells change during biofilm formation and that the EPS proteins function as non-specific adhesions during biofilm formation. The physicochemical traits of the cell surface and the EPS proteins give us an insight into the forces that drive biofilm formation and maintenance in B. cereus.     

Persister cells in a biofilm treated with a biocide

This study investigated the physiology and behaviour following treatment with ortho-phthalaldehyde (OPA), of Pseudomonas fluorescens in both the planktonic and sessile states. Steady-state biofilms and planktonic cells were collected from a bioreactor and their extracellular polymeric substances (EPS) were extracted using a method that did not destroy the cells. Cell structure and physiology after EPS extraction were compared in terms of respiratory activity, morphology, cell protein and polysaccharide content, and expression of the outer membrane proteins (OMP). Significant differences were found between the physiological parameters analysed. Planktonic cells were more metabolically active, and contained greater amounts of proteins and polysaccharides than biofilm cells. Moreover, biofilm formation promoted the expression of distinct OMP. Additional experiments were performed with cells after EPS extraction in order to compare the susceptibility of planktonic and biofilm cells to OPA. Cells were completely inactivated after exposure to the biocide (minimum bactericidal concentration, MBC = 0.55 ± 0.20 mM for planktonic cells; MBC = 1.7 ± 0.30 mM for biofilm cells). After treatment, the potential of inactivated cells to recover from antimicrobial exposure was evaluated over time. Planktonic cells remained inactive over 48 h while cells from biofilms recovered 24 h after exposure to OPA, and the number of viable and culturable cells increased over time. The MBC of the recovered biofilm cells after a second exposure to OPA was 0.58 ± 0.40 mM, a concentration similar to the MBC of planktonic cells. This study demonstrates that persister cells may survive in biocide-treated biofilms, even in the absence of EPS.     

Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms

Exopolymeric substances (EPS) are important for biofilm formation and their chemical composition may influence biofilm properties. To explore these relationships the chemical composition of EPS from Bacillus subtilis NCIB 3610 biofilms grown in sucrose-rich (SYM) and sucrose-poor (MSgg and Czapek) media was studied. We observed marked differences in composition of EPS polymers isolated from all three biofilms or from spent media below the biofilms. The polysaccharide levan dominated the EPS of SYM grown biofilms, while EPS from biofilms grown in sucrose-poor media contained significant amounts of proteins and DNA in addition to polysaccharides. The EPS polymers differed also in size with very large polymers (Mw>2000 kDa) found only in biofilms, while small polymers (Mw<200 kD) dominated in the EPS isolated from spent media. Biofilms of the eps knockout were significantly thinner than those of the tasA knockout in all media. The biofilm defective phenotypes of tasA and eps mutants were, however, partially compensated in the sucrose-rich SYM medium. Sucrose supplementation of Czapek and MSgg media increased the thickness and stability of biofilms compared to non-supplemented controls. Since sucrose is essential for synthesis of levan and the presence of levan was confirmed in all biofilms grown in media containing sucrose, this study for the first time shows that levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of B. subtilis floating biofilms. In addition, we propose that this polysaccharide, when incorporated into the biofilm EPS, may also serve as a nutritional reserve.     

Relevance of Polymeric Matrix Enzymes During Biofilm Formation

Extracellular polymeric substances (EPS) contribute to biofilm stability and adhesion properties. The EPS matrix might also be a site for free extracellular enzyme activity; however, little is known about participation of enzyme activity in EPS during biofilm formation. In this study, we analyzed the activities of beta-glucosidase, leu-aminopeptidase, and beta-glucosaminidase during the colonization of artificial substrata (glass tiles) in a stream distinguishing enzyme activity in EPS matrix (matrix-enzymes) and total biofilm extracellular enzyme activity. The 1-h incubation of a biofilm suspension and cation-exchange resin followed by centrifugation seems appropriate to extract the matrix fraction (supernatant) and measure matrix enzymes (including free and linked to EPS) in freshwater biofilms, although there is a methodological limitation for using a biofilm suspension instead of an undisrupted biofilm. Total biofilm activities and matrix-enzyme activities showed similar capabilities to decompose organic matter compounds, with a greater capacity for peptide decomposition (leu-aminopeptidase) than for polysaccharides (beta-glucosidase), and a low decomposition of chitin and peptidoglycan (beta-glucosaminidase). Matrix-enzyme activity increased with colonization time, but more slowly than that of total enzyme activity. At the beginning of the colonization experiment (days 1-4) matrix enzymes accounted for 65-81% of total biofilm enzyme activity. Higher proportion of polysaccharides in EPS versus total biofilm, and higher matrix-enzyme activities per microgram of polysaccharides in the EPS were measured during the first 1-3 days of biofilm formation, indicating a high rate of enzyme release into the matrix during this period. Relative contribution of matrix-enzyme activities decreased as biofilm matures, but was maintained at 13-37% of total enzyme activity at the 42- to 49-day-old biofilm. These enzymes, retained and conserved in the EPS, may contribute to community metabolism. When analyzing extracellular enzymes in biofilms, the contribution of matrix enzymes must be considered, especially for young biofilms.     

Extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms: characterization by infrared spectroscopy and proteomics

The composition of extracellular polymeric substances (EPS) from Shewanella sp. HRCR-1 biofilms was investigated using infrared spectroscopy and proteomics to provide insight into potential ecophysiological functions and redox activity of the EPS. Both bound and loosely associated EPS were extracted from Shewanella sp. HRCR-1 biofilms prepared using a hollow-fibre membrane biofilm reactor. Fourier transform infrared spectra revealed the presence of proteins, polysaccharides, nucleic acids, membrane lipids and fatty acids in the EPS fractions. Using a global proteomic approach, a total of 58 extracellular and outer membrane proteins were identified in the EPS. These included homologues of multiple Shewanella oneidensis MR-1 proteins that potentially contribute to key physiological biofilm processes, such as biofilm-promoting protein BpfA, surface-associated serine protease, nucleotidases (CpdB and UshA), an extracellular lipase, and oligopeptidases (PtrB and a M13 family oligopeptidase lipoprotein). In addition, 20 redox proteins were found in extracted EPS. Among the detected redox proteins were the homologues of two S. oneidensis MR-1 c-type cytochromes, MtrC and OmcA, which have been implicated in extracellular electron transfer. Given their detection in the EPS of Shewanella sp. HRCR-1 biofilms, c-type cytochromes may contribute to the possible redox activity of the biofilm matrix and play important roles in extracellular electron transfer reactions.     

Evidence of compositional differences between the extracellular and intracellular DNA of a granular sludge biofilm

Aims: Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules. Methods and Results: Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR‐based denaturing gradient‐gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA. Conclusions: Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm. Significance and Impact of the Study: The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms.     

Extraction of extracellular polymeric substances from extreme acidic microbial biofilms

The efficiency of five extraction methods for extracellular polymeric substances (EPS) was compared on three benthic eukaryotic biofilms isolated from an extreme acidic river, Río Tinto (SW, Spain). Three chemical methods (MilliQ water, NaCl, and ethylenediamine tetraacetic acid [EDTA]) and two physical methods (Dowex 50.8 and Crown Ether cation exchange resins) were tested. The quality and quantity of the EPS extracted from acidic biofilms varied according to which EPS extraction protocol was used. Higher amounts were obtained when NaCl and Crown Ether resins were used as extractant agents, followed by EDTA, Dowex, and MilliQ. EPS amounts varied from approximately 155 to 478 mg g⁻¹ of dry weight depending on the extraction method and biofilm analyzed. EPS were primarily composed of carbohydrate, heavy metals, and humic acid, plus small quantities of proteins and DNA. Neutral hexose concentrations corresponded to more than 90% of the total EPS dry weight. The proportions of each metals in the EPS extracted with EDTA are similar to the proportions present in the water from each locality where the biofilms were collected except for Al, Cu, Zn, and Pb. In this study, the extracellular matrix heavy metal sorption efficiencies of five methods for extracting EPS from eukaryotic acidic biofilms were compared.     

Potential role of bacterial extracellular polymeric substances as biosorbent material for arsenic bioremediation

Carcinogenic effects of arsenic through consumption of contaminated water are an alarming threat and there is an emergent need to reduce extremely high levels of toxic arsenic from environment. Bacterial biofilms produce polyanionic extracellular polymeric substance (EPS) that is considered an excellent biosorbent material for the remediation of toxic metals and metalloids. This study was aimed to investigate the role of bacterial EPS in arsenic bioremediation. EPS was extracted from biofilm forming and arsenic reducer bacterial strains that were isolated from industrial waste water and characterized biochemically. Fourier transform infrared spectroscopy was also performed to study functional groups. Both Exiguobacterium profundum PT2 and Ochrobactrum ciceri SW1 exhibited enhanced EPS production in the presence of arsenic. Arsenic stress increased protein and carbohydrate contents in the EPS of both bacterial strains as indicated by the peaks of 1363 to 1613 and 1035 to 1218?cm^?1 wavenumbers, respectively to cope with arsenic present in the surroundings. Shifting of peaks in As⁵⁺ treated samples from 1363 to 1379, 847 to 800 and 1211 to 1134?cm^?1 demonstrated the involvement of proteins, carbohydrates and phosphates in the sequestration of arsenic. Scanning electron microscopic examination of EPS revealed structural alterations such as the presence of closely embedded large clumps with interstitial spaces between stacked layers of the EPS of E. profundum PT2 treated with As⁵⁺ displayed the enhanced polysaccharide content and arsenic sorption. Therefore, increased production of bacterial EPS with large number of polyanionic functional groups on its surface having tendency to sequester arsenic through electrostatic or covalent interactions presented EPS an excellent biosorbent material for arsenic bioremediation.     

Antibiofilm activity and mode of action of DMSO alone and its combination with afatinib against Gram-negative pathogens

Biofilms are complex microbial communities that tend to attach to either biotic or abiotic surface. Enclosed in a self-produced extracellular polymeric substance (EPS) matrix, the biofilms often cause persistent infections. The objective of this study was to investigate the antibiofilm activity of dimethyl sulfoxide (DMSO) and afatinib against Gram-negative pathogens. Test microorganisms used in this study were Escherichia coli ATCC 1299, Pseudomonas aeruginosa ATCC 10145, and Salmonella typhimurium ATCC 14028. Biofilms were developed in 96-well microplate at 37°C for 24 h. Following removal of non-adherent cells, analysis of biofilm viability, biofilm biomass, and extracellular polymeric substances (EPS) matrix were performed using resazurin assay, crystal violet assay, and attenuated total reflectance fourier transform infrared (ATR-FTIR) spectroscopy, respectively. Bradford protein assay was conducted to determine the total amount of EPS proteins. The results demonstrated that both 32% DMSO alone and its combination with 3.2 μg/mL afatinib were effective in killing biofilm cells and reducing biofilm biomass. IR spectral variations of EPS matrix of biofilms in the range between 1700 and 900 cm^?1 were also observed. Reduction in EPS proteins verified the chemical modifications of EPS matrix. In conclusion, 32% DMSO alone and its combination with 3.2 μg/mL afatinib showed remarkable antibiofilm activities against Gram-negative pathogens. It was suggested that the biofilm inhibition was mediated by the chemical modification of EPS matrix.