Process modelling for industrial scale polyhydroxybutyrate production using fructose, formic acid and CO2: Assessing carbon sources and economic viability.

Polyhydroxybutyrate (PHB) is a biodegradable polymer that has potential to replace petroleum-derived plastics. However, the commercialisation of PHB is hindered by high production costs. In this study, the material flow and economics of an industrial scale PHB production process using fructose, formic acid and carbon dioxide (CO2) as carbon sources were simulated and analysed. The lowest breakeven price of 3.64 $/kg PHB was obtained when fructose was utilized as carbon source. When formic acid and CO2 were used, the breakeven price was 10.30 and 10.24 $/kg PHB due to raw material cost, respectively. Although using formic acid and CO2 is more expensive, they meet the emerging sustainable needs for plastic production and contribute to the circular economy via CO2 fixation. This study suggests that the use of formic acid and CO2 as feedstock for PHB production has potential to become competitive in the bioplastic market with further research.

Investigating Mass Transfer and Reaction Engineering Characteristics in a Membrane Biofilm Using Cupriavidus necator H16.

Membrane biofilm reactors are a growing trend in wastewater treatment whereby gas-transfer membranes provide efficient bubbleless aeration. Recently, there has been a growing interest in using these bioreactors for industrial biotechnology using microorganisms that can metabolise gaseous substrates. Since gas fermentation is limited by the low solubilities of gaseous substrates in liquid media, it is critical to characterise mass transfer rates of gaseous substrates to enable the design of membrane biofilm reactors. The objective of this study is to measure and analyse mass transfer rates and reaction engineering characteristics for a single tube membrane biofilm reactor using Cupriavidus necator H16. At elevated Reynolds numbers, the dominant resistance for gas diffusion shifts from the liquid boundary layer to the membrane. The biofilm growth rate was observed to decrease after 260 µm at 96 h. After 144 h, some sloughing of the biofilm occurred. Oxygen uptake rate and substrate utilisation rate for the biofilm developed showed that the biofilm changes from a single-substrate limited regime to a dual-substrate-limited regime after 72 h which alters the localisation of the microbial activity within the biofilm. This study shows that this platform technology has potential applications for industrial biotechnology.

Interaction between Engineered Pluronic Silica Nanoparticles and Bacterial Biofilms: Elucidating the Role of Nanoparticle Surface Chemistry and EPS Matrix.

Nanoparticles (NPs) are considered a promising tool in the context of biofilm control. Many studies have shown that different types of NPs can interfere with the bacterial metabolism and cellular membranes, thus making them potential antibacterial agents; however, fundamental understanding is still lacking on the exact mechanisms involved in these actions. The development of NP-based approaches for effective biofilm control also requires a thorough understanding of how the chosen nanoparticles will interact with the biofilm itself, and in particular with the biofilm self-produced extracellular polymeric matrix (EPS). This work aims to provide advances in the understanding of the interaction between engineered fluorescent pluronic silica (PluS) nanoparticles and bacterial biofilms, with a main focus on the role of the EPS matrix in the accumulation and diffusion of the particles in the biofilm. It is demonstrated that particle surface chemistry has a key role in the different lateral distribution and specific affinity to the biofilm matrix components. The results presented in this study contribute to our understanding of biofilm-NP interactions and promote the principle of the rational design of smart nanoparticles as an important tool for antibiofilm technology.

A polyhydroxyalkanoates bioprocess improvement case study based on four fed-batch feeding strategies.

The modelling and optimization of a process for the production of the medium chain length polyhydroxyalkanoate (mcl-PHA) by the bacterium Pseudomonas putida KT2440 when fed a synthetic fatty acid mixture (SFAM) was investigated. Four novel feeding strategies were developed and tested using a constructed model and the optimum one implemented in further experiments. This strategy yielded a cell dry weight of 70.6 g l-1 in 25 h containing 38% PHA using SFAM at 5 l scale. A phosphate starvation strategy was implemented to improve PHA content, and this yielded 94.1 g l-1 in 25 h containing 56% PHA using SFAM at 5 l scale. The process was successfully operated at 20 l resulting in a cell dry weight of 91.2 g l-1 containing 65% PHA at the end of a 25-h incubation.

Membrane bioreactors for the production of value-added products: Recent developments, challenges and perspectives.

The potential of membrane bioreactors to produce value-added products such as biofuels, biopolymers, proteins, organic acids and lipids at high productivities is emerging. Despite the promising results at laboratory scale, industrial deployment of this technology is hindered due to challenges associated with scale-up. This review aims to address these challenges and create a framework to encourage further research directed towards industrial application of membrane bioreactors to produce value-added products. This review describes the current state-of-the art in such bioreactor systems by exploiting membranes to increase the mass transfer rate of the limiting substrates, reach high cell concentrations and separate the inhibitory substances that may inhibit the bioconversion reaction. It also covers the current trends in commercialization, challenges linked with membrane usage, such as high costs and membrane fouling, and proposes possible future directions for the wider application of membrane bioreactors.

Biomimetic Polymer Surfaces by High Resolution Molding of the Wings of Different Cicadas.

Recent studies have shown that insect wings have evolved to have micro- and nanoscale structures on the wing surface, and biomimetic research aims to transfer such structures to application-specific materials. Herein, we describe a simple and cost-effective method of replica molding the wing topographies of four cicada species using UV-curable polymers. Different polymer blends of polyethylene glycol diacrylate and polypropylene glycol diacrylate were used as molding materials and a molding chamber was designed to precisely control the x, y, and z dimensions. Analysis by scanning electron microscopy showed that structures ranged from 148 to 854 nm in diameter, with a height range of 191-2368 nm, and wing patterns were transferred with high fidelity to the crosslinked polymer. Finally, bacterial cell studies show that the wing replicas possess the same antibacterial effect as the cicada wing from which they were molded. Overall, this work shows a quick and simple method for patterning UV-curable polymers without the use of expensive equipment, making it a highly accessible means of producing microstructured materials with biological properties.

Synthesis and self-assembly of curcumin-modified amphiphilic polymeric micelles with antibacterial activity.

BACKGROUND: The ubiquitous nature of bacterial biofilms combined with the enhanced resistance towards antimicrobials has led to the development of an increasing number of strategies for biofilm eradication. Such strategies must take into account the existence of extracellular polymeric substances, which obstruct the diffusion of antibiofilm agents and assists in the maintenance of a well-defended microbial community. Within this context, nanoparticles have been studied for their drug delivery efficacy and easily customised surface. Nevertheless, there usually is a requirement for nanocarriers to be used in association with an antimicrobial agent; the intrinsically antimicrobial nanoparticles are most often made of metals or metal oxides, which is not ideal from ecological and biomedical perspectives. Based on this, the use of polymeric micelles as nanocarriers is appealing as they can be easily prepared using biodegradable organic materials. RESULTS: In the present work, micelles comprised of poly(lactic-co-glycolic acid) and dextran are prepared and then functionalised with curcumin. The effect of the functionalisation in the micelle's physical properties was elucidated, and the antibacterial and antibiofilm activities were assessed for the prepared polymeric nanoparticles against Pseudomonas spp. cells and biofilms. It was found that the nanoparticles have good penetration into the biofilms, which resulted in enhanced antibacterial activity of the conjugated micelles when compared to free curcumin. Furthermore, the curcumin-functionalised micelles were efficient at disrupting mature biofilms and demonstrated antibacterial activity towards biofilm-embedded cells. CONCLUSION: Curcumin-functionalised poly(lactic-co-glycolic acid)-dextran micelles are novel nanostructures with an intrinsic antibacterial activity tested against two Pseudomonas spp. strains that have the potential to be further exploited to deliver a secondary bioactive molecule within its core.

Enzyme-Functionalized Mesoporous Silica Nanoparticles to Target Staphylococcus aureus and Disperse Biofilms.

BACKGROUND: Staphylococcus aureus biofilms pose a unique challenge in healthcare due to their tolerance to a wide range of antimicrobial agents. The high cost and lengthy timeline to develop novel therapeutic agents have pushed researchers to investigate the use of nanomaterials to deliver antibiofilm agents and target biofilm infections more efficiently. Previous studies have concentrated on improving the efficacy of antibiotics by deploying nanoparticles as nanocarriers. However, the dispersal of the extracellular polymeric substance (EPS) matrix in biofilm-associated infections is also critical to the development of novel nanoparticle-based therapies. METHODS: This study evaluated the efficacy of enzyme-functionalized mesoporous silica nanoparticles (MSNs) against methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) biofilms. MSNs were functionalized with the enzyme lysostaphin, which causes cell lysis of S. aureus bacteria. This was combined with two other enzyme functionalized MSNs, serrapeptase and DNase I which will degrade protein and eDNA in the EPS matrix, to enhance eradication of the biofilm. Cell viability after treatment with enzyme-functionalized MSNs was assessed using a MTT assay and CLSM, while crystal violet staining was used to assess EPS removal. RESULTS: The efficacy of all three enzymes against S. aureus cells and biofilms was significantly improved when they were immobilized onto MSNs. Treatment efficacy was further enhanced when the three enzymes were used in combination against both MRSA and MSSA. Regardless of biofilm maturity (24 or 48 h), near-complete dispersal and killing of MRSA biofilms were observed after treatment with the enzyme-functionalized MSNs. Disruption of mature MSSA biofilms with a polysaccharide EPS was less efficient, but cell viability was significantly reduced. CONCLUSION: The combination of these three enzymes and their functionalization onto nanoparticles might extend the therapeutic options for the treatment of S. aureus infections, particularly those with a biofilm component.

Electric Field Tunability of Photoluminescence from a Hybrid Peptide-Plasmonic Metal Microfabricated Chip.

Enhancement of fluorescence through the application of plasmonic metal nanostructures has gained substantial research attention due to the widespread use of fluorescence-based measurements and devices. Using a microfabricated plasmonic silver nanoparticle-organic semiconductor platform, we show experimentally the enhancement of fluorescence intensity achieved through electro-optical synergy. Fluorophores located sufficiently near silver nanoparticles are combined with diphenylalanine nanotubes (FFNTs) and subjected to a DC electric field. It is proposed that the enhancement of the fluorescence signal arises from the application of the electric field along the length of the FFNTs, which stimulates the pairing of low-energy electrons in the FFNTs with the silver nanoparticles, enabling charge transport across the metal-semiconductor template that enhances the electromagnetic field of the plasmonic nanoparticles. Many-body perturbation theory calculations indicate that, furthermore, the charging of silver may enhance its plasmonic performance intrinsically at particular wavelengths, through band-structure effects. These studies demonstrate for the first time that field-activated plasmonic hybrid platforms can improve fluorescence-based detection beyond using plasmonic nanoparticles alone. In order to widen the use of this hybrid platform, we have applied it to enhance fluorescence from bovine serum albumin and Pseudomonas fluorescens. Significant enhancement in fluorescence intensity was observed from both. The results obtained can provide a reference to be used in the development of biochemical sensors based on surface-enhanced fluorescence.

Demand response through reject water scheduling in water resource recovery facilities: A demonstration with BSM2.

The objective of this paper is to determine the importance of integrating peak demand mitigation and future energy pricing structures for process modelling of conventional water resource recovery facilities (WRRFs) when evaluating energy cost and control strategies. The well-established benchmark simulation model (BSM2) is used to monitor energy usage, and a detailed holistic study of different flow streams is performed in order to establish potential opportunities for flexible control of WRRF energy demand. Secondly, a detailed framework is introduced to optimize scheduling control strategies for the reject water stream while considering peak electricity demand avoidance as well as completing a comprehensive energy cost model based on current and anticipated future energy tariff structures. The reject water scheduling strategies, without other active controls (e.g. aeration), revealed 63.4% average peak demand mitigation and €10,755 cumulative annual energy cost savings on a 100k population equivalent WRRF without a deterioration in effluent quality. Analysis of different reject water scheduling control strategies shows that reject water scheduling can be an effective tool for energy cost optimisation under alternative electricity tariff structures. These strategies also deliver electricity peak demand mitigation.

Tailoring Nanoparticle-Biofilm Interactions to Increase the Efficacy of Antimicrobial Agents Against Staphylococcus aureus.

BACKGROUND: Considering the timeline required for the development of novel antimicrobial drugs, increased attention should be given to repurposing old drugs and improving antimicrobial efficacy, particularly for chronic infections associated with biofilms. Methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) are common causes of biofilm-associated infections but produce different biofilm matrices. MSSA biofilm cells are typically embedded in an extracellular polysaccharide matrix, whereas MRSA biofilms comprise predominantly of surface proteins and extracellular DNA (eDNA). Nanoparticles (NPs) have the potential to enhance the delivery of antimicrobial agents into biofilms. However, the mechanisms which influence the interactions between NPs and the biofilm matrix are not yet fully understood. METHODS: To investigate the influence of NPs surface chemistry on vancomycin (VAN) encapsulation and NP entrapment in MRSA and MSSA biofilms, mesoporous silica nanoparticles (MSNs) with different surface functionalization (bare-B, amine-D, carboxyl-C, aromatic-A) were synthesised using an adapted Stöber method. The antibacterial efficacy of VAN-loaded MSNs was assessed against MRSA and MSSA biofilms. RESULTS: The two negatively charged MSNs (MSN-B and MSN-C) showed a higher VAN loading in comparison to the positively charged MSNs (MSN-D and MSN-A). Cellular binding with MSN suspensions (0.25 mg mL-1) correlated with the reduced viability of both MSSA and MRSA biofilm cells. This allowed the administration of low MSNs concentrations while maintaining a high local concentration of the antibiotic surrounding the bacterial cells. CONCLUSION: Our data suggest that by tailoring the surface functionalization of MSNs, enhanced bacterial cell targeting can be achieved, leading to a novel treatment strategy for biofilm infections.

Predicting wastewater treatment plant performance during aeration demand shifting with a dual-layer reaction settling model.

Demand response (DR) programmes encourage energy end users to adjust their consumption according to energy availability and price. Municipal wastewater treatment plants are suitable candidates for the application of such programmes. Demand shedding through aeration control, subject to maintaining the plant operational limits, could have a large impact on the plant DR potential. Decreasing the aeration intensity may promote the settling of the particulate components present in the reactor mixed liquor. The scope of this study is thus to develop a mathematical model to describe this phenomenon. For this purpose, Benchmark Simulation Model No.1 was extended by implementing a dual-layer settling model in one of the aerated tanks and combining it with biochemical reaction kinetic equations. The performance of this extended model was assessed in both steady-state and dynamic conditions, switching the aeration system off for 1 hour during each day of simulation. This model will have applications in the identification of potential benefits and issues related to DR events, as well as in the simulation of the plant operation where aerated tank settling is implemented.

Interactions between functionalised silica nanoparticles and Pseudomonas fluorescens biofilm matrix: A focus on the protein corona.

Biofilms are microbial communities embedded in an extracellular polymeric matrix and display an enhanced tolerance to the action of antimicrobials. The emergence of novel functionalised nanoparticles is considered a promising avenue for the development of biofilm-specific antimicrobial technologies. However, there is a gap in the understanding of interactions between nanoparticles and the biofilm matrix. Particularly, questions are raised on how nanoparticle charge and surface groups play a role in aggregation when in contact with biofilm components. Herein we present the synthesis of four types of silica nanoparticles and undertake an analysis of their interactions with Pseudomonas fluorescens biofilm matrix. The effect of the biofilm matrix components on the charge and aggregation of the nanoparticles was assessed. Additionally, the study focused on the role of matrix proteins, with the in-depth characterisation of the protein corona of each nanoparticle by Liquid Chromatography with Tandem Mass Spectrometry experiments. The protein corona composition is dependent on the nanoparticle type; non-functionalised nanoparticles show less protein selectivity, whereas carboxylate-functionalised nanoparticles prefer proteins with a higher isoelectric point. These outcomes provide insights into the field of biofilm-nanoparticle interactions that can be valuable for the design of new nano-based targeting systems in future anti-biofilm applications.

A high throughput method to investigate nanoparticle entrapment efficiencies in biofilms.

The commercial use of nanoparticles has increased in recent years due to their unique characteristics, including high surface area, modifiable shape and surface charge and size-dependent properties. Consequently, a greater number of nanomaterials are now being released into the environment and inevitably interact with the natural ecosystem. Bacterial biofilms have the potential to capture and retain nanoparticles, however the factors determining the specific nanoparticle entrapment efficiencies of biofilms are not yet fully understood. Based on fluorescent intensity measurements we developed a simple and straightforward method that allowed the entrapment of different silica nanoparticles by two Pseudomonas strains to be quantified. It was determined that, regardless of nanoparticle size or surface functionalisation, Pseudomonas putida biofilms showed enhanced entrapment efficiencies compared to Pseudomonas fluorescens biofilms. It was also noted that both biofilms showed a higher entrapment capacity towards positively charged NPs. The method developed has the potential to be utilized for high throughput biofilm screening studies in order to develop a new understating of the relationship between nanoparticle characteristics and its uptake by bacterial biofilms.

Benchmarking leachate co-treatment strategies in municipal wastewater treatment plants under dynamic conditions and energy prices.

Combined leachate treatment at municipal wastewater treatment plants (WWTPs) is applicable to a certain extent depending on the leachate composition, treatment plant configuration and its capacity. Co-treatment of leachate at WWTPs has several advantages, but due to increasingly stringent discharge standards applied in WWTPs, it has become more problematic. This study was undertaken to investigate the impact of leachate feeding strategies on effluent quality and the aeration energy costs of WWTPs. A modified version of Benchmark Simulation Model No.1 was used to develop, test and compare several leachate feeding and WWTP control strategies in the context of dynamic pollutant loads and energy prices. The results highlighted that combined leachate treatment led to a deterioration in the quality of discharged wastewater, as indicated by a 12-20% increase in effluent quality index. Additionally, it adversely affected aeration energy demand and cost of the plant by increasing them 1.7-2.3% and 0.8-2.5%, respectively. The impacts could be mitigated by adjusting leachate flow based on effluent ammonium concentrations and by using advanced process control, i.e. feedback ammonium control for dissolved oxygen regulation in aerobic reactors. The study demonstrates that modeling can be used as a valuable tool to assess the potential impacts of leachate co-treatment and develop better management strategies.

Surface functionalization-dependent localization and affinity of SiO2 nanoparticles within the biofilm EPS matrix.

The contribution of the biofilm extracellular polymeric substance (EPS) matrix to reduced antimicrobial susceptibility in biofilms is widely recognised. As such, the direct targeting of the EPS matrix is a promising biofilm control strategy that allows for the disruption of the matrix, thereby allowing a subsequent increase in susceptibility to antimicrobial agents. To this end, surface-functionalized nanoparticles (NPs) have received considerable attention. However, the fundamental understanding of the interactions occurring between engineered NPs and the biofilm EPS matrix has not yet been fully elucidated. An insight into the underlying mechanisms involved when a NP interacts with the EPS matrix will aid in the design of more efficient NPs for biofilm control. Here we demonstrate the use of highly specific fluorescent probes in confocal laser scanning microscopy (CLSM) to illustrate the distribution of EPS macromolecules within the biofilm. Thereafter, a three-dimensional (3D) colocalization analysis was used to assess the affinity of differently functionalized silica NPs (SiNPs) and EPS macromolecules from Pseudomonas fluorescens biofilms. Results show that both the charge and surface functional groups of SiNPs dramatically affected the extent to which SiNPs interacted and localized with EPS macromolecules, including proteins, polysaccharides and DNA. Hypotheses are also presented about the possible physicochemical interactions which may be dominant in EPS matrix-NP interactions. This research not only develops an innovative CLSM-based methodology for elucidating biofilm-nanoparticle interactions but also provides a platform on which to build more efficient NP systems for biofilm control.

Enhancing curcumin's solubility and antibiofilm activity via silica surface modification.

Bacterial biofilms are microbial communities in which bacterial cells in sessile state are mechanically and chemically protected against foreign agents, thus enhancing antibiotic resistance. The delivery of active compounds to the inside of biofilms is often hindered due to the existence of the biofilm extracellular polymeric substances (EPS) and to the poor solubility of drugs and antibiotics. A possible strategy to overcome the EPS barrier is the incorporation of antimicrobial agents into a nanocarrier, able to penetrate the matrix and deliver the active substance to the cells. Here, we report the synthesis of antimicrobial curcumin-conjugated silica nanoparticles (curc-NPs) as a possibility for dealing with these issues. Curcumin is a known antimicrobial agent and to overcome its low solubility in water it was grafted onto the surface of silica nanoparticles, the latter functioning as nanocarrier for curcumin into the biofilm. Curc-NPs were able to impede the formation of model P. putida biofilms up to 50% and disrupt mature biofilms up to 54% at 2.5 mg mL-1. Cell viability of sessile cells in both cases was also considerably affected, which is not observed for curcumin delivered as a free compound at the same concentration. Furthermore, proteomics of extracted EPS matrix of biofilms grown in the presence of free curcumin and curc-NPs revealed differences in the expression of key proteins related to cell detoxification and energy production. Therefore, curc-NPs are presented here as an alternative for curcumin delivery that can be exploited not only to other bacterial strains but also to further biological applications.

Nanoparticle-Biofilm Interactions: The Role of the EPS Matrix.

The negative consequences of biofilms are widely reported. A defining feature of biofilms is the extracellular matrix, a complex mixture of biomacromolecules, termed EPS, which contributes to reduced antimicrobial susceptibility. EPS targeting is a promising, but underexploited, approach to biofilm control allowing disruption of the matrix and thereby increasing the susceptibility to antimicrobials. Nanoparticles (NPs) can play a very important role as 'carriers' of EPS matrix disruptors, and several approaches have recently been proposed. In this review, we discuss the application of nanoparticles as antibiofilm technologies with a special emphasis on the role of the EPS matrix in the physicochemical regulation of the nanoparticle-biofilm interaction. We highlight the use of nanoparticles as a platform for a new generation of antibiofilm approaches.

Ratiometric Imaging of the in Situ pH Distribution of Biofilms by Use of Fluorescent Mesoporous Silica Nanosensors.

Biofilms are communities of microorganisms enclosed in a self-generated matrix of extracellular polymeric substances. While biofilm recalcitrance and persistence are caused by several factors, a reduction in antimicrobial susceptibility has been closely associated with the generation of pH gradients within the biofilm structure. Cells embedded within the biofilm create a localized acidic microenvironment, which is unaffected by the external pH. Therefore, pH monitoring is a promising approach for understanding the complexities of a three-dimensional heterogeneous biofilm. A fluorescent pH nanosensor was designed through the synthesis of mesoporous silica nanoparticles (47 ± 5 nm diameter) conjugated to a pH-sensitive dye (fluorescein) and a pH-insensitive dye (rhodamine B) as an internal standard (dye-MSNs). The fluorescence intensity of fluorescein (IF) reduced significantly as the pH was decreased from 8.5 to 3.5. In contrast, the fluorescence intensity of rhodamine B (IR) remained constant at any pH. The ratio of IF/IR produced a sigmoidal curve with respect to the pH, in a working pH range between 4.5 and 7.5. Dye-MSNs enabled the measurement of pH gradients within Pseudomonas fluorescens WCS 365 biofilm microcolonies. The biofilms showed spatially distinct low-pH regions that were enclosed into large clusters corresponding to high-cell-density areas. Also present were small low-pH areas that spread indistinctly throughout the microcolony caused by the mass transfer effect. The lowest detected pH within the inner core of the microcolonies was 5.1, gradually increasing to a neutral pH toward the exterior of the microcolonies. The dye-MSNs were able to fully penetrate the biofilm matrix and allowed a quantitative ratiometric analysis of pH gradients and distribution throughout the biofilm, which was independent of the nanoparticle concentration.

Simultaneous oxidation of ammonium and tetracycline in a membrane aerated biofilm reactor.

The membrane aerated biofilms reactor (MABR) is an emerging technology in wastewater treatment with particular advantages including high rate nitrification, and very high oxygen transfer efficiencies. In this study a synthetic feed water incorporating tetracycline (TC) was investigated in a MABR. Simultaneous removal of ammonium and tetracycline (TC) in the reactor, formation of TC transformation products (TPs), and microbial community analysis in the biofilm growing on the membrane were performed. A range of TC and ammonium loading rates and the effect of different intra-membrane oxygen pressures were on treatment performance were systematically investigated. Successful nitrification and TC degradation were achieved with the highest TC removal (63%) obtained at a HRT of 18 h HRT and 0.41 bar gas pressure. It has shown that different operating conditions (HRT and gas pressure) do not cause a significant change in ammonium removal. The concentration of TPs such as ETC, EATC, and ATC was determined to be at the ppb level. Molecular results showed that MABR reactor was mainly dominated by ß-proteobacteria. The relative abundance of this group decreased in parallel with the increasing ammonium and TC loading.