Polysaccharide differences between planktonic and biofilm-associated EPS from Pseudomonas fluorescens B52

The polysaccharides associated with free (planktonic) and surface-attached (biofilm) cells from cultures of Pseudomonas fluorescens strain B52 were compared. Variations in the attached matrix due to surface material (glass or stainless steel) were also analyzed. Two digestion methods were used to optimize the recoveries of sugars, uronic acids and acidic substituents. The yield of analyzable material after digestion reached 90% for the material associated to the biofilms, though only 20–30% for that bound to planktonic cells. The polysaccharide(s) in the biofilm had glucuronic and guluronic acids as main components, besides rhamnose, glucose and glucosamine. The proportion of glucuronic to guluronic acid was higher in the polysaccharide(s) found in biofilms formed on stainless steel than in those on glass.     

delta-Aminolaevulinic acid mediated photodynamic antimicrobial chemotherapy on Pseudomonas aeruginosa planktonic and biofilm cultures

To demonstrate photodynamic antimicrobial chemotherapy (PACT) against planktonic and biofilm cultures of Pseudomonas aeruginosa, using photoporphyrin IX which could be endogenously synthesized by administrating delta-aminolaevulinic acid (delta-ALA), and a light emitted diode (LED) array to photoactivate the photosensitizer. P. aeruginosa suspended cells or biofilms, grown on a rotating disk reactor, were treated by different concentrations of delta-ALA in the dark for 1 h, followed by LED irradiation for various time. Regrowth experiments were conducted by placed PACT-treated disks back to a sterile reactor. Viable cells were determined by serial dilution and plate counts. Both P. aeruginosa planktonic and biofilm cells were inhibited by PACT with light doses or photosensitizer concentrations increasing. Treatments of planktonic cells with 10 mM delta-ALA and incident dose 240 J cm(-2) or 7.5 mM ALA and incident dose 360 J cm(-2) led to completely photoinactivation. No viable biofilm cells were found after treatment of 20 mM delta-ALA and incident dose 240 J cm(-2). However, regrowth was observed once PACT-treated biofilms were put back to a sterile reactor. Regrowth could be prevented only if biofilm samples were treated PACT twice. delta-ALA-mediated PACT on P. aeruginosa planktonic and biofilm cells was effective, though the detailed mechanism still required further investigation.     

Effects of photoactivated titanium dioxide nanopowders and coating on planktonic and biofilm growth of Pseudomonas aeruginosa

We exploited the ability of photocatalytic titanium dioxide (TiO(2)) as an agent for the biofilm control. Two photocatalytic systems were investigated: a 3 g L(-1) suspension of TiO(2) nanopowder in demineralized water and glass slides coated with a TiO(2) thin film, achieved by sol-gel deposition. A running protocol for the photoactivation of TiO(2) was set up using the dye rhodamine B. The microorganisms studied were Pseudomonas stutzeri, Pseudomonas aeruginosa and a Bacillus cereus-group as planktonic cells. P. aeruginosa biofilms were also studied at both the solid-liquid and the solid-air interface. The TiO(2) nanopowder produced 1-log reduction of Bacillus sp. planktonic cells in 24 h, 2-log reduction of P. stutzeri planktonic cells in 30 min and 1-log reduction of P. aeruginosa planktonic cells in 2 h compared with non-photo-activated TiO(2). TiO(2) thin film produced almost a complete eradication of P. aeruginosa planktonic cells (initial concentration 10(8) cells mL(-1)) in 24 h compared to a 3-log reduction caused by UV-A light alone. In contrast, neither the photocatalytic treatment with TiO(2) film nor that with TiO(2) nanopowder had any effect on P. aeruginosa biofilms at all the interfaces investigated. Possible explanations for these findings, and for the discrepancy between this work and literature data, are discussed.     

The influence of nickel on the adhesion ability of Desulfovibrio desulfuricans

The build-up of biofilms on metals surfaces may lead to severe corrosion, especially in the presence of sulphate-reducing bacteria (SRB). To prevent the deterioration of material caused by biofilms it is necessary to understand the processes governing biofilm development including mechanisms of cell adhesion. Additionally, corrosion of metallic surfaces due to bacteria may lead to the dissolution of metallic elements that may further affect adhesion and biofilm development. A study was carried out to evaluate how the presence of nickel in the substrata affects the adhesion ability of Desulfovibrio desulfuricans. The substrata tested were stainless steel 304 (SS), metallic nickel (Ni) and polymethylmetacrylate (PMMA), a non-metallic material used as control. The influence of nickel on SRB growth and its relation to adhesion was also checked. A statistically significant difference in the number of adhered cells to the materials tested was detected, with higher bacterial number on nickel, followed by SS and finally by PMMA. The higher number of SRB adhered to steel compared with PMMA may be explained by differences in hydrophobicity, in roughness and in the electron-acceptor character of the substrata. Additionally, bacterial growth was found to be positively affected by the presence of nickel as revealed by a significant increase in the specific growth rate of SRB in the presence of increased nickel concentrations.     

Efflux Pump Inhibitor Potentiates Antimicrobial Photodynamic Inactivation of Enterococcus faecalis Biofilm

Microbial biofilm architecture contains numerous protective features, including extracellular polymeric material that render biofilms impermeable to conventional antimicrobial agents. This study evaluated the efficacy of antimicrobial photodynamic inactivation (aPDI) of Enterococcus faecalis biofilms. The ability of a cationic, phenothiazinium photosensitizer, methylene blue (MB) and an anionic, xanthene photosensitizer, rose bengal (RB) to inactivate biofilms of E. faecalis (OG1RF and FA 2-2) and disrupt the biofilm structure was evaluated. Bacterial cells were tested as planktonic suspensions, intact biofilms and biofilm-derived suspensions obtained by the mechanical disruption of biofilms. The role of a specific microbial efflux pump inhibitor (EPI), verapamil hydrochloride in the MB-mediated aPDI of E. faecalis biofilms was also investigated. The results showed that E. faecalis biofilms exhibited significantly higher resistance to aPDI when compared with E. faecalis in suspension (P < 0.001). aPDI with cationic MB produced superior inactivation of E. faecalis strains in a biofilm along with significant destruction of biofilm structure when compared with anionic RB (P < 0.05). The ability to inactivate biofilm bacteria was further enhanced when the EPI was used with MB (P < 0.001). These experiments demonstrated the advantage of a cationic phenothiazinium photosensitizer combined with an EPI to inactivate biofilm bacteria and disrupt biofilm structure.     

Evaluation of epifluorescence image analysis of biofilm growth on stainless steel surfaces

Biofilm growth of Bacillus subtilis, Pseudomonas fragi, Pediococcus inopinatus and Listeria monocytogenes was studied on stainless steel surfaces at room and low temperatures to evaluate the results of traditional hygiene measures. The results were compared with those of image analysis of stainless steel surfaces in an epifluorescence microscope. Statistical analyses were carried out to determine the variations between the conventional cultivation swab method, the glycocalyx amount obtained using swabbing, and the values of the areas of the biofilm, slime and cells. As a general rule, old biofilms showed total counts at approximately the same levels as the young biofilm. The results showed that temperature affected the results for all strains except B. subtilis. The strains of Pe. inopinatus and Ps. fragi showed increased attachment at 6°C and L. monocytogenes at 25°C. The biofilm slime was more easily detached than the cells. The results indicated that the traditional swab method is not reliable for the measurement of biofilm formation on surfaces.     

Photodynamic Inactivation of Bacterial and Yeast Biofilms With a Cationic Porphyrin

The efficiency of 5,10,15,20‐tetrakis(1‐methylpyridinium‐4‐yl)porphyrin tetra‐iodide (Tetra‐Py⁺‐Me) in the photodynamic inactivation of single‐species biofilms of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans and mixed biofilms of S. aureus and C. albicans was evaluated. The effect on the extracellular matrix of P. aeruginosa was also assessed. Irradiation with white light up to an energy dose of 64.8 J cm^?2 in the presence of 20 μm of Tetra‐Py⁺‐Me caused significant inactivation in all single‐species biofilms (3–6 log reductions), although the susceptibility was attenuated in relation to planktonic cells. In mixed biofilms, the inactivation of S. aureus was as efficient as in single‐species biofilms but the susceptibility of C. albicans decreased. In P. aeruginosa biofilms, a reduction of 81% in the polysaccharide content of the matrix was observed after treatment with a 20 μm PS concentration and a total light dose of 64.8 J cm^?2. The results show that the Tetra‐Py⁺‐Me causes significant inactivation of the microorganisms, either in biofilms or in the planktonic form, and demonstrate that polysaccharides of the biofilm matrix may be a primary target of photodynamic damage.     

Effects of operating conditions on the adhesive strength of Pseudomonas fluorescens biofilms in tubes

Understanding the mechanical properties of biofilms, especially the force required to disrupt them and remove them from substrata is very important to development of antibiofouling strategies. In this work, a novel micromanipulation technique with a specially designed T-shaped probe has been developed to serve as an experimental means to measure directly the adhesive strength of biofouling deposits on the surface of a glass test stud. The basic principle of this novel technique is to pull away a whole biofilm accumulated on the surface of a glass test stud with T-shaped probe, and to measure simultaneously the force imposed on the biofilm. The adhesive strength between the biofilms and the surface to which they are attached, is defined as the work per unit area required to remove the biofilms from the surface. The biofouling experiments were performed on an elaborate design of a simulated heat exchanger system. A monoculture of Pseudomonas fluorescens was chosen as the fouling microorganism for the laboratory studies. Results indicate that the adhesive strength of the biofilm was affected by the conditions of operation, such as biofilm age, nutrient concentration, suspended cell concentration, pH, surface roughness of the substratum and fluid velocity. As noted, the effect of fluid velocity on the biofilm adhesive strength seemed to overwhelm other factors. At the same operating conditions, the biofilm adhesive strength increased as the fluid velocity increased within the range of 0.6-1.6m/s. In addition, the flow-related biofilm structures were observed that biofilms generally grew as a more compact pattern at the higher fluid velocity. Apparently, the fluid velocity can affect the biofilm structure, which in turn determines the biofilm adhesive strength. The knowledge of the biofilm adhesive strength with associated influences of the operating conditions may be used to define better cleaning procedures.     

Physicochemical Characterization of Acidiphilium sp. Biofilms

The biofilm formation of a strain of the extremophile bacterium Acidiphilium sp., capable of donating electrons directly to electrodes, was studied by different surface characterization techniques. We develop a method that allows the simultaneous study of bacterial biofilms by means of fluorescence microscopy and atomic force microscopy (AFM), in which transparent graphitic flakes deposited on a glass substrate are used as a support for the biofilm. The majority of the cells present on the surface were viable, and the growth of the biofilms over time showed a critical increase of the extracellular polymeric substances (EPS) as well as the formation of nanosized particles inside the biofilm. Also, the presence of Fe in Acidiphilium biofilms was determined by X‐ray photoelectron spectroscopy (XPS), whereas surface‐enhanced infrared absorption spectroscopy indicated the presence of redox‐active proteins.     

Small molecule control of bacterial biofilms

Bacterial biofilms are defined as a surface attached community of bacteria embedded in a matrix of extracellular polymeric substances that they have produced. When in the biofilm state, bacteria are more resistant to antibiotics and the host immune response than are their planktonic counterparts. Biofilms are increasingly recognized as being significant in human disease, accounting for 80% of bacterial infections in the body and diseases associated with bacterial biofilms include: lung infections of cystic fibrosis patients, colitis, urethritis, conjunctivitis, otitis, endocarditis and periodontitis. Additionally, biofilm infections of indwelling medical devices are of particular concern, as once the device is colonized infection is virtually impossible to eradicate. Given the prominence of biofilms in infectious diseases, there has been an increased effort toward the development of small molecules that will modulate bacterial biofilm development and maintenance. In this review, we highlight the development of small molecules that inhibit and/or disperse bacterial biofilms through non-microbicidal mechanisms. The review discuses the numerous approaches that have been applied to the discovery of lead small molecules that mediate biofilm development. These approaches are grouped into: (1) the identification and development of small molecules that target one of the bacterial signaling pathways involved in biofilm regulation, (2) chemical library screening for compounds with anti-biofilm activity, and (3) the identification of natural products that possess anti-biofilm activity, and the chemical manipulation of these natural products to obtain analogues with increased activity.     

Use of hydrophilic hydroxypropyl methacrylate/ethylene glycol methacrylate packing material in size-exclusion chromatography

Spherical particles of hydroxypropyl methacrylate/ethylene glycol methacrylate copolymer were synthesized in-house for use in size-exclusion chromatography. The porous hydrophilic material was packed in glass and stainless steel columns to evaluate their chromatographic performance. The support particles were small (approximately 20 A), and the average pore size was in the low range of mesopores (approximately 100 A). The packed columns were calibrated by using polysaccharide dextrans, showing a good range of separation for molecular weights between 10000 and 600000 daltons. The packing material appears to separate the large molecules through the size-exclusion mechanism. Polysaccharides and polypeptides dissolved in adequate mobile phases were injected into the packed column. The separation of the macromolecules was consistent with the size-exclusion mechanism. Application of the packing material to the separation of small molecules (alkyl alcohols) was also investigated.     

Spring constants and adhesive properties of native bacterial biofilm cells measured by atomic force microscopy

Bacterial biofilms were imaged by atomic force microscopy (AFM), and their elasticity and adhesion to the AFM tip were determined from a series of tip extension and retraction cycles. Though the five bacterial strains studied included both Gram-negative and -positive bacteria and both environmental and laboratory strains, all formed simple biofilms on glass surfaces. Cellular spring constants, determined from the extension portion of the force cycle, varied between 0.16+/-0.01 and 0.41+/-0.01 N/m, where larger spring constants were measured for Gram-positive cells than for Gram-negative cells. The nonlinear regime in the extension curve depended upon the biomolecules on the cell surface: the extension curves for the smooth Gram-negative bacterial strains with the longest lipopolysaccharides on their surface had a larger nonlinear region than the rough bacterial strain with shorter lipopolysaccharides on the surface. Adhesive forces between the retracting silicon nitride tip and the cells varied between cell types in terms of the force components, the distance components, and the number of adhesion events. The Gram-negative cells' adhesion to the tip showed the longest distance components, sometimes more than 1 microm, whereas the shortest distance adhesion events were measured between the two Gram-positive cell types and the tip. Fixation of free-swimming planktonic cells by NHS and EDC perturbed both the elasticity and the adhesive properties of the cells. Here we consider the biochemical meaning of the measured physical properties of simple biofilms and implications to the colonization of surfaces in the first stages of biofilm formation.     

Approaches for Mitigating Microbial Biofilm-Related Drug Resistance: A Focus on Micro- and Nanotechnologies

Biofilms play an essential role in chronic and healthcare-associated infections and are more resistant to antimicrobials compared to their planktonic counterparts due to their (1) physiological state, (2) cell density, (3) quorum sensing abilities, (4) presence of extracellular matrix, (5) upregulation of drug efflux pumps, (6) point mutation and overexpression of resistance genes, and (7) presence of persister cells. The genes involved and their implications in antimicrobial resistance are well defined for bacterial biofilms but are understudied in fungal biofilms. Potential therapeutics for biofilm mitigation that have been reported include (1) antimicrobial photodynamic therapy, (2) antimicrobial lock therapy, (3) antimicrobial peptides, (4) electrical methods, and (5) antimicrobial coatings. These approaches exhibit promising characteristics for addressing the impending crisis of antimicrobial resistance (AMR). Recently, advances in the micro- and nanotechnology field have propelled the development of novel biomaterials and approaches to combat biofilms either independently, in combination or as antimicrobial delivery systems. In this review, we will summarize the general principles of clinically important microbial biofilm formation with a focus on fungal biofilms. We will delve into the details of some novel micro- and nanotechnology approaches that have been developed to combat biofilms and the possibility of utilizing them in a clinical setting.     

Surface morphology and surface energy of anode materials influence power outputs in a multi-channel mediatorless bio-photovoltaic (BPV) system

Bio-photovoltaic cells (BPVs) are a new photo-bio-electrochemical technology for harnessing solar energy using the photosynthetic activity of autotrophic organisms. Currently power outputs from BPVs are generally low and suffer from low efficiencies. However, a better understanding of the electrochemical interactions between the microbes and conductive materials will be likely to lead to increased power yields. In the current study, the fresh-water, filamentous cyanobacterium Pseudanabaena limnetica (also known as Oscillatoria limnetica) was investigated for exoelectrogenic activity. Biofilms of P. limnetica showed a significant photo response during light-dark cycling in BPVs under mediatorless conditions. A multi-channel BPV device was developed to compare quantitatively the performance of photosynthetic biofilms of this species using a variety of different anodic conductive materials: indium tin oxide-coated polyethylene terephthalate (ITO), stainless steel (SS), glass coated with a conductive polymer (PANI), and carbon paper (CP). Although biofilm growth rates were generally comparable on all materials tested, the amplitude of the photo response and achievable maximum power outputs were significantly different. ITO and SS demonstrated the largest photo responses, whereas CP showed the lowest power outputs under both light and dark conditions. Furthermore, differences in the ratios of light?:?dark power outputs indicated that the electrochemical interactions between photosynthetic microbes and the anode may differ under light and dark conditions depending on the anodic material used. Comparisons between BPV performances and material characteristics revealed that surface roughness and surface energy, particularly the ratio of non-polar to polar interactions (the CQ ratio), may be more important than available surface area in determining biocompatibility and maximum power outputs in microbial electrochemical systems. Notably, CP was readily outperformed by all other conductive materials tested, indicating that carbon may not be an optimal substrate for microbial fuel cell operation.     

Dünnschichtchromatographische trennung und bestimmung von uronsäuren und uronsäurelactonen

A new method for the separation of galacturonic, glucuronic, mannuronic and guluronic acid and the corresponding lactones by thin-layer chromatography is described. The determination of mixtures of galacturonic and glucuronic acid (1–50 μg) is described. Neutral monosaccharides up to loo μg and lower concentrations of inor ganic materials do not interfere with the separation.     

Electrochemical characterization of anodic biofilms enriched with glucose and acetate in single-chamber microbial fuel cells

This study used a simple and efficient electrochemical technique, cyclic voltammogram (CV), to quantitatively measure the electron transfer capability of anodic biofilms enriched with acetate and glucose in single-chamber microbial fuel cells (MFCs). Two pairs of distinct redox peaks were observed by CV measurements in both biofilms, identical to the CV features of a pure Geobacter strain. The CVs also revealed a higher density of electroactive species in the acetate-enriched biofilm than that in the glucose-enriched biofilm. Based on the scan rate analysis, the apparent electron transfer rate constants (k(app)) in the acetate-enriched biofilm and glucose-enriched biofilm were determined to be 0.82 and 0.15s(-1), respectively, which supported the higher power output of the MFC fed with acetate. Meanwhile, the pH dependence of the biofilms was studied by monitoring the changes of the biofilm redox peak currents and potentials. It is concluded that redox reaction of the electrochemical active species in biofilms is pH dependent, and both electrons and protons are involved in the redox reactions.     

Xanthene Dyes and Green LED for the Inactivation of Foodborne Pathogens in Planktonic and Biofilm States

This study evaluated the rose bengal‐ and erythrosine‐mediated photoinactivation against Salmonella Typhimurium and Staphylococcus aureus planktonic and sessile cells using green LED as a light source. The free‐living or 2‐day‐old biofilm cells were treated with different concentrations of the photosensitizing agents and subjected to irradiation. Only 5 min photosensitization with rose bengal at 25 nmol L^?1 and 75 μmol L^?1 completely eliminated S. aureus and S. Typhimurium planktonic cells, respectively. Erythrosine at 500 nmol L^?1 and 5 min of light exposure also reduced S. aureus planktonic cells to undetectable levels. Eradication of S. aureus biofilms was achieved when 500 μmol L^?1 of erythrosine or 250 μmol L^?1 of rose bengal was combined with 30 min of irradiation. Scanning electron microscopy allowed the observation of morphological changes in planktonic cells and disruption of the biofilm architecture after photodynamic treatment. The overall data demonstrate that rose bengal and erythrosine activated by green LED may be a targeted strategy for controlling foodborne pathogens in both planktonic and sessile states.     

Shear-flow induced detachment of Saccharomyces cerevisiae from stainless steel: influence of yeast and solid surface properties

The present study focused on the shear-induced detachment of Saccharomyces cerevisiae in adhesive contact with a 316L stainless steel surface using a shear stress flow chamber, with a view to determining the respective influence of the yeast surface properties and the support characteristics. The effect of cultivation of S. cerevisiae yeast cells on their subsequent detachment from the solid surface was particularly investigated. In order to elucidate the role of stainless steel, non-metallic supports were used as control, covering a broad range of surface properties such as surface free energy and roughness: polypropylene (hydrophobic), polystyrene (mildly hydrophobic, similar to stainless steel) and glass (hydrophilic). All materials were very smooth with respect to the size of yeast. First, experiments were carried out on two types of yeast cells, just rehydrated in saline solution, a biological model widely used in the literature. The influence of the ionic strength (1.5 and 150 mM NaCl) on glass and stainless steel was evaluated. Unlike on glass, no clear evidence was found for electrostatic repulsion with stainless steel since high adhesion was observed whatever the ionic strength. A lack of correlation in adhesion results was also obtained when considering the surface physico-chemical characteristics of type I (hydrophilic) and type II (hydrophobic) rehydrated cells and those of both polymers. It was postulated that unavoidable “sticky” compounds were present on the cell wall, which could not be completely removed during the successive washings of the rehydrated cell suspension before use. This could dramatically alter the yeast surface properties and modify the adhesion strength, thus clearly demonstrating the necessity to work with yeast coming from fresh cultures. Biologically active yeast cells were then used. Once cultured, type I- and type II-yeast cells were shown to exhibit the same hydrophilic properties. Regardless of the material used, for the same ionic strength (150 mM NaCl), yeast adhesion was drastically reduced compared to rehydrated yeast cells. Among all the materials tested, the specificity of 316L stainless steel was clearly established. Indeed, for glass and polymers, cell adhesion was substratum-dependent and driven by the balance between the Lifshitz-van der Waals and Lewis acid/base interactions. Despite nearly identical surface free energies for polystyrene and stainless steel, the metallic surface promoted a totally distinct behaviour which was characterized by a strong – although highly variable – yeast adhesion.     

Analysis of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1 Biofilm Protein Profile After Exposure to <Emphasis Type="Italic">n</Emphasis>-Butanolic <Emphasis Type="Italic">Cyclamen coum</Emphasis> Extract Alone and in Combination with Ciprofloxacin

Pseudomonas aeruginosa biofilm-related infections are the major cause of premature death in cystic fibrosis patients. Strategies to induce biofilm dispersal are of interest, because of their potential in preventing biofilm-related infections. Our previous work demonstrated that n-butanolic Cyclamen coum extract with ciprofloxacin could eliminate 1- and 3-day-old P. aeruginosa PAO1 biofilms. To gain new insights into the role of C. coum extract and its synergistic effect with ciprofloxacin in eliminating P. aeruginosa PAO1 biofilms, two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry-based protein identification were used. Changes in the bacterial protein expression were analyzed when 3-day-old biofilm cells were exposed to the C. coum extract alone and in combination with ciprofloxacin. Proteins involved in alginate biosynthesis, quorum sensing, adaptation/protection, carbohydrate and amino acid metabolism showed a weaker expression in the C. coum extract-ciprofloxacin-treated biofilm cells compared to those in the untreated cells. Interestingly, the proteome of C. coum extract-ciprofloxacin-treated biofilm revealed more resemblance to the planktonic phenotype than to the biofilm phenotype. It appears that saponin extract in combination with ciprofloxacin causes biofilm disruption due to several mechanisms such as motility induction, cell envelope integrity perturbation, stress protein expression reduction, and more importantly, signal transduction perturbation. In conclusion, exposure to a combination of biofilm dispersal such as saponin extract and antimicrobial agents may offer a novel strategy to control preestablished, persistent P. aeruginosa biofilms and biofilm-related infections.     

Upgrading Solid Digestate from Anaerobic Digestion of Agricultural Waste as Performance Enhancer for Starch-Based Mulching Biofilm

Developing a green and sustainable method to upgrade biogas wastes into high value-added products is attracting more and more public attention. The application of solid residues as a performance enhancer in the manufacture of biofilms is a prospective way to replace conventional plastic based on fossil fuel. In this work, solid digestates from the anaerobic digestion of agricultural wastes, such as straw, cattle and chicken manures, were pretreated by an ultrasonic thermo-alkaline treatment to remove the nonfunctional compositions and then incorporated in plasticized starch paste to prepare mulching biofilms by the solution casting method. The results indicated that solid digestate particles dispersed homogenously in the starch matrix and gradually aggregated under the action of a hydrogen bond, leading to a transformation of the composites to a high crystalline structure. Consequently, the composite biofilm showed a higher tensile strength, elastic modulus, glass transition temperature and degradation temperature compared to the pure starch-based film. The light, water and GHG (greenhouse gas) barrier properties of the biofilm were also reinforced by the addition of solid digestates, performing well in sustaining the soil quality and minimizing N₂O or CH₄ emissions. As such, recycling solid digestates into a biodegradable plastic substitute not only creates a new business opportunity by producing high-performance biofilms but also reduces the environmental risk caused by biogas waste and plastics pollution.