Analytical methods for quantifying PS and PVC Nanoplastic attachment to activated sludge Bacteria and their impact on community structure.

Nanoplastics are anticipated to be ubiquitous in various environmental compartments. However, challenges in analytical methods hinder our understanding of risks related to specific nanplastics characteristics such as size and chemical compositions, and interactions between nanoplastics and microorganisms. In this study, we applied fit-for-purpose analytical methods and techniques to understand how nanoplastic chemical composition influences their interaction with bacteria collected from activated sludge. When exposed to polystyrene (PS) and polyvinyl chloride (PVC) nanoplastics for 5 days, the nanoplastics attached to the bacteria. Specifically, on day 1, there was a significant predominance of PS nanoplastics over PVC ones of similar size and shape, possibly due to differences in their chemical composition. After 5 days, there is a substantial decrease in nanoplastics attached to bacteria, suggesting bacterial defence mechanisms may reduce particles attachment over time. The overall bacterial community structure demonstrated a high degree of resilience. This resilience highlights the ability of microbial communities to maintain their structure despite nanoplastic stressors, as evidenced by consistent alpha diversity, PCoA, and PERMANOVA results. Understanding these mechanisms is crucial for assessing nanoplastic fate and thus environmental impacts.

Mechanistic Insights into the Biological Effects of Engineered Nanomaterials: A Focus on Gold Nanoparticles.

Nanotechnology has great potential to significantly advance the biomedical field for the benefit of human health. However, the limited understanding of nano-bio interactions leading to unknowns about the potential adverse health effects of engineered nanomaterials and to the poor efficacy of nanomedicines has hindered their use and commercialization. This is well evidenced considering gold nanoparticles, one of the most promising nanomaterials for biomedical applications. Thus, a fundamental understanding of nano-bio interactions is of interest to nanotoxicology and nanomedicine, enabling the development of safe-by-design nanomaterials and improving the efficacy of nanomedicines. In this review, we introduce the advanced approaches currently applied in nano-bio interaction studies-omics and systems toxicology-to provide insights into the biological effects of nanomaterials at the molecular level. We highlight the use of omics and systems toxicology studies focusing on the assessment of the mechanisms underlying the in vitro biological responses to gold nanoparticles. First, the great potential of gold-based nanoplatforms to improve healthcare along with the main challenges for their clinical translation are presented. We then discuss the current limitations in the translation of omics data to support risk assessment of engineered nanomaterials.

Early stage biofilm formation on bio-based microplastics in a freshwater reservoir.

Bio-based plastics (BP) produced from renewable biomass resources, such as high-density polyethylene (HDPE), polylactic acid (PLA) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), is currently increasing in terms of both products and applications. However, their biodegradability and environmental fate are not yet fully understood, especially in freshwaters. Here, we present the results of an in-situ study in a freshwater reservoir, where we submerged HDPE, PLA and PHBV microscale BP (mBP) in dialysis bags to enable exchange of small organic and inorganic molecules, including nutrients, with the surrounding water. After one and two months, the bacterial biofilm that formed on each mBP was characterised by 16S rRNA amplicon sequencing. After two-months, Oxalobacteraceae, Pedosphaeraceae, Flavobacteriaceae (Flavobacterium) and Chitinophagaceae (Ferruginibacter) had increased by up to four times. Both these and other common members (≥1 % relative total biomass) of the microbial community were similarly abundant on all mBP. Low-abundance (0.3-1 %) bacterial taxa, however, were significantly more diverse and differed on each mBP. Notably, some low-abundance families and genera increased on specific materials, e.g. Sphingomonadaceae on HDPE, Sphingobacteriaceae on PHBV, Gemmatimonas and Crenothrix on PLA. Overall, abundant bacteria were regarded as a pioneering community, while low-abundance bacteria were more diverse and preferred mBP types in the early stages of biofilm formation on mBP. It could be influenced by the environmental conditions, where nutrient levels and low temperatures might shape the low-abundance of attached bacterial communities than the plastic material itself.

Growth suppression of bacteria by biofilm deterioration using silver nanoparticles with magnetic doping.

Decades of antibiotic use and misuse have generated selective pressure toward the rise of antibiotic-resistant bacteria, which now contaminate our environment and pose a major threat to humanity. According to the evolutionary "Red queen theory", developing new antimicrobial technologies is both urgent and mandatory. While new antibiotics and antibacterial technologies have been developed, most fail to penetrate the biofilm that protects bacteria against external antimicrobial attacks. Hence, new antimicrobial formulations should combine toxicity for bacteria, biofilm permeation ability, biofilm deterioration capability, and tolerability by the organism without renouncing compatibility with a sustainable, low-cost, and scalable production route as well as an acceptable ecological impact after the ineluctable release of the antibacterial compound in the environment. Here, we report on the use of silver nanoparticles (NPs) doped with magnetic elements (Co and Fe) that allow standard silver antibacterial agents to perforate bacterial biofilms through magnetophoretic migration upon the application of an external magnetic field. The method has been proved to be effective in opening micrometric channels and reducing the thicknesses of models of biofilms containing bacteria such as Enterococcus faecalis, Enterobacter cloacae, and Bacillus subtilis. Besides, the NPs increase the membrane lipid peroxidation biomarkers through the formation of reactive oxygen species in E. faecalis, E. cloacae, B. subtilis, and Pseudomonas putida colonies. The NPs are produced using a one-step, scalable, and environmentally low-cost procedure based on laser ablation in a liquid, allowing easy transfer to real-world applications. The antibacterial effectiveness of these magnetic silver NPs may be further optimized by engineering the external magnetic fields and surface conjugation with specific functionalities for biofilm disruption or bactericidal effectiveness.

Gum Hydrocolloids Reinforced Silver Nanoparticle Sponge for Catalytic Degradation of Water Pollutants.

The accumulation of organic contaminants including dyes in aquatic systems is of significant environmental concern, necessitating the development of affordable and sustainable materials for the treatment/elimination of these hazardous pollutants. Here, a green synthesis strategy has been used to develop a self-assembled gum kondagogu-sodium alginate bioconjugate sponge adorned with silver nanoparticles, for the first time. The properties of the nanocomposite sponge were then analyzed using FTIR, TGA, SEM, and MicroCT. The ensued biobased sponge exhibited hierarchical microstructure, open cellular pores, good shape memory, and mechanical properties. It merges the attributes of an open cellular porous structure with metal nanoparticles and are envisaged to be deployed as a sustainable catalytic system for reducing contaminants in the aqueous environment. This nanocomposite sponge showed enhanced catalytic effectiveness (km values up to 37 min-1 g-1 and 44 min-1 g-1 for methylene blue and 4-nitrophenol, respectively), antibacterial properties, reusability, and biodegradability (65% biodegradation in 28 days).

Dissolved iron released from nanoscale zero-valent iron (nZVI) activates the defense system in bacterium Pseudomonas putida, leading to high tolerance to oxidative stress.

Nanoscale zero-valent iron (nZVI) has increasingly been applied to remediate aquifers polluted by organochlorines or heavy metals. As a result, bacteria in the vicinity of remediate action can be stressed by surplus iron released from nZVI. However, the understanding of the iron stress defense pathways during this process is currently incomplete. Therefore, we aimed to elucidate the physiological and transcriptomic response of the bacterium, Pseudomonas putida NCTC 10936, to 100 mg/L of nZVI and 44.5 µg/L of dissolved iron obtained from nZVI suspension. Cell viability was neither affected by nZVI nor dissolved iron, although the dissolved iron caused stress that altered the cell physiology and caused the generation of smaller cells, whereas cells were elongated in the presence of nZVI. Transcriptomic analysis confirmed the observed stronger physiological effect caused by dissolved iron (in total 3839 differentially expressed genes [DEGs]) than by nZVI (945 DEGs). Dissolved iron (but not nZVI) activated genes involved in oxidative stress-related pathways, antioxidant activity, carbohydrate and energy metabolism, but downregulated genes associated with flagellar assembly proteins and two-component systems involved in sensing external stimuli. As a result, bacteria very effectively faced oxidative insults and cell viability was not affected.

Enhanced degradation of sulfamethoxazole by a modified nano zero-valent iron with a ß-cyclodextrin polymer: Mechanism and toxicity evaluation.

Rising concern about emerging and already persisting pollutants in water has urged the scientific community to develop novel remedial techniques. A new group of remediation methods is based on the modification of nanoscale zero-valent iron particles (nZVI), which are well known for treating volatile organic compounds and heavy metals. The properties of nZVI may be further enhanced by modifying their structure or surface using "green" polymers. Herein, nZVI was modified by a ß-cyclodextrin polymer (ß-CDP), which is considered an environmentally safe and inexpensive adsorbent of contaminants. This composite was used for the first time for the degradation of sulfamethoxazole (SMX). Coating by ß-CDP not only enhanced the degradation of SMX (>95%, under 10 min) by the nanoparticles in a wide pH range (3-9) and enabled their efficient reusability (for three cycles) but also made the coated nZVI less toxic to the model bioindicator microalga Raphidocelis subcapitata. Moreover, degradation products of SMX were found to be less toxic to Escherichia coli bacteria and R. subcapitata microalga, contrary to the SMX antibiotic itself, indicating a simple and eco-friendly cleaning process. This research aims to further stimulate and develop novel remedial techniques based on nZVI, and provides a potential application in the degradation of antibiotics in a wide pH range. Moreover, the wealth of available cyclodextrin materials used for surface modification may open a way to discover more efficient and attractive composites for environmental applications.

Alkenyl succinic anhydride modified tree-gum kondagogu: A bio-based material with potential for food packaging.

Tree gums are a class of abundantly available carbohydrate polymers that have not been explored thoroughly in film fabrication for food packaging. Films obtained from pristine tree gums are often brittle, hygroscopic, and lack mechanical strength. This study focuses on the chemical modification of gum kondagogu using long-chain alkenyl groups of dodecenyl succinic anhydride (DDSA), an esterifying agent that introduces a 12-carbon hydrophobic chain to the kondagogu structure. The esterification reaction was confirmed by 1H nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The effect of nano-cellulose as an additive on various film properties was investigated. The developed films were characterized for their mechanical, morphological, optical, barrier, antibacterial, and biodegradable properties. The inclusion of long-chain carbon groups acted as internal plasticizers and resulted in an amorphous structure with better film-forming ability, improved hydrophobicity, and higher elongation at break values. The modified films exhibited antibacterial properties and excellent biodegradability under aerobic conditions.

Discovering the potential of an nZVI-biochar composite as a material for the nanobioremediation of chlorinated solvents in groundwater: Degradation efficiency and effect on resident microorganisms.

Abiotic and biotic remediation of chlorinated ethenes (CEs) in groundwater from a real contaminated site was studied using biochar-based composites containing nanoscale zero-valent iron (nZVI/BC) and natural resident microbes/specific CE degraders supported by a whey addition. The material represented by the biochar matrix decorated by isolated iron nanoparticles or their aggregates, along with the added whey, was capable of a stepwise dechlorination of CEs. The tested materials (nZVI/BC and BC) were able to decrease the original TCE concentration by 99% in 30 days. Nevertheless, regarding the transformation products, it was clear that biotic as well as abiotic transformation mechanisms were involved in the transformation process when nonchlorinated volatiles (i.e., methane, ethane, ethene, and acetylene) were detected after the application of nZVI/BC and nZVI/BC with whey. The whey addition caused a massive increase in bacterial biomass in the groundwater samples (monitored by 16S rRNA sequencing and qPCR) that corresponded with the transformation of trichloro- and dichloro-CEs, and this process was accompanied by the formation of less chlorinated products. Moreover, the biostimulation step also eliminated the adverse effect caused by nZVI/BC (decrease in microbial biomass after nZVI/BC addition). The nZVI/BC material or its aging products, and probably together with vinyl chloride-respiring bacteria, were able to continue the further reductive dechlorination of dichlorinated CEs into nonhalogenated volatiles. Overall, the results of the present study demonstrate the potential, feasibility, and environmental safety of this nanobioremediation approach.

Impact of Zero-Valent Iron on Freshwater Bacterioplankton Metabolism as Predicted from 16S rRNA Gene Sequence Libraries.

The application of zero-valent iron particles (ZVI) for the treatment of heavily polluted environment and its biological effects have been studied for at least two decades. Still, information on the impact on bacterial metabolic pathways is lacking. This study describes the effect of microscale and nanoscale ZVI (mZVI and nZVI) on the abundance of different metabolic pathways in freshwater bacterial communities. The metabolic pathways were inferred from metabolism modelling based on 16S rRNA gene sequence data using paprica pipeline. The nZVI changed the abundance of numerous metabolic pathways compared to a less influencing mZVI. We identified the 50 most affected pathways, where 31 were related to degradation, 17 to biosynthesis, and 2 to detoxification. The linkage between pathways was two times higher in nZVI samples compared to mZVI, and was specifically higher considering the arsenate detoxification II pathway. Limnohabitans and Roseiflexus were linked to the same pathways in both nZVI and mZVI. The prediction of metabolic pathways increases our knowledge of the impacts of nZVI and mZVI on freshwater bacterioplankton.

An across-species comparison of the sensitivity of different organisms to Pb-based perovskites used in solar cells.

Organic-inorganic perovskite solar cells (PSCs) are promising candidates as photovoltaic cells. Recently, they have attracted significant attention due to certified power conversion efficiencies exceeding 23%, low-cost engineering, and superior electrical/optical characteristics. These PSCs extensively utilize a perovskite-structured composite with a hybrid of Pb-based nanomaterials. Operation of them may cause the release of Pb-based nanoparticles. However, limited information is available regarding the potential toxicity of Pb-based PSCs on various organisms. This study conducted a battery of in vitro and in vivo toxicity bioassays for three quintessential Pb-based PSCs (CH3NH3PbI3, NHCHNH3PbBr3, and CH3NH3PbBr3) using progressively more complex forms of life. For all species tested, the three different perovskites had comparable toxicities. The viability of Caco-2/TC7 cells was lower than that of A549 cells in response to Pb-based PSC exposure. Concentration-dependent toxicity was observed for the bioluminescent bacterium Vibrio fischeri, for soil bacterial communities, and for the nematode Caenorhabditis elegans. Neither of the tested Pb-based PSCs particles had apparent toxicity to Pseudomonas putida. Among all tested organisms, V. fischeri showed the highest sensitivity with EC50 values (30 min of exposure) ranging from 1.45 to 2.91 mg L-1. Therefore, this study recommends that V. fischeri should be preferably utilized to assess. PSC toxicity due to its increased sensitivity, low costs, and relatively high throughput in a 96-well format, compared with the other tested organisms. These results highlight that the developed assay can easily predict the toxic potency of PSCs. Consequently, this approach has the potential to promote the implementation of the 3Rs (Replacement, Reduction, and Refinement) principle in toxicology and decrease the dependence on animal testing when determining the safety of novel PSCs.

Green Synthesis of Metal and Metal Oxide Nanoparticles and Their Effect on the Unicellular Alga Chlamydomonas reinhardtii.

Recently, the green synthesis of metal nanoparticles has attracted wide attention due to its feasibility and very low environmental impact. This approach was applied in this study to synthesise nanoscale gold (Au), platinum (Pt), palladium (Pd), silver (Ag) and copper oxide (CuO) materials in simple aqueous media using the natural polymer gum karaya as a reducing and stabilising agent. The nanoparticles' (NPs) zeta-potential, stability and size were characterised by Zetasizer Nano, UV-Vis spectroscopy and by electron microscopy. Moreover, the biological effect of the NPs (concentration range 1.0-20.0 mg/L) on a unicellular green alga (Chlamydomonas reinhardtii) was investigated by assessing algal growth, membrane integrity, oxidative stress, chlorophyll (Chl) fluorescence and photosystem II photosynthetic efficiency. The resulting NPs had a mean size of 42 (Au), 12 (Pt), 1.5 (Pd), 5 (Ag) and 180 (CuO) nm and showed high stability over 6 months. At concentrations of 5 mg/L, Au and Pt NPs only slightly reduced algal growth, while Pd, Ag and CuO NPs completely inhibited growth. Ag, Pd and CuO NPs showed strong biocidal properties and can be used for algae prevention in swimming pools (CuO) or in other antimicrobial applications (Pd, Ag), whereas Au and Pt lack these properties and can be ranked as harmless to green alga.

In vitro and environmental toxicity of reduced graphene oxide as an additive in automotive lubricants.

Despite the ground-breaking potential of nanomaterials, their safe and sustainable incorporation into an array of industrial markets prompts a deep and clear understanding of their potential toxicity for both humans and the environment. Among the many materials with great potential, graphene has shown promise in a variety of applications; however, the impact of graphene based products on living systems remains poorly understood. In this paper, we illustrate that via exploiting the tribological properties of graphene nanosheets, we can successfully improve both the frictional behaviour and the anti-wear capacity of lubricant oil for mechanical transmission. By virtue of reducing friction and enhancing lubricant lifetimes, we can forecast a reduction in friction based energy loss, in addition to a decrease in the carbon footprint of vehicles. The aforementioned positive environmental impact is further strengthened considering the lack of acute toxicity found in our extensive in vitro investigation, in which both eukaryotic and prokaryotic cells were tested. Collectively, our body of work suggests that by the use of safe nanoadditives we could contribute to reducing the environmental impact of transportation and therein take a positive step towards a more sustainable automotive sector. The workflow proposed here for the evaluation of human and environmental toxicity will allow for the study of nanosized bare graphene material and can be broadly applied to the translation of graphene-based nanomaterials into the market.

Biological effects of four iron-containing nanoremediation materials on the green alga Chlamydomonas sp.

As nanoremediation strategies for in-situ groundwater treatment extend beyond nanoiron-based applications to adsorption and oxidation, ecotoxicological evaluations of newly developed materials are required. The biological effects of four new materials with different iron (Fe) speciations ([i] FerMEG12 - pristine flake-like milled Fe(0) nanoparticles (nZVI), [ii] Carbo-Iron® - Fe(0)-nanoclusters containing activated carbon (AC) composite, [iii] Trap-Ox® Fe-BEA35 (Fe-zeolite) - Fe-doped zeolite, and [iv] Nano-Goethite - 'pure' FeOOH) were studied using the unicellular green alga Chlamydomonas sp. as a model test system. Algal growth rate, chlorophyll fluorescence, efficiency of photosystem II, membrane integrity and reactive oxygen species (ROS) generation were assessed following exposure to 10, 50 and 500 mg L-1 of the particles for 2 h and 24 h. The particles had a concentration-, material- and time-dependent effect on Chlamydomonas sp., with increased algal growth rate after 24 h. Conversely, significant intracellular ROS levels were detected after 2 h, with much lower levels after 24 h. All Fe-nanomaterials displayed similar Z-average sizes and zeta-potentials at 2 h and 24 h. Effects on Chlamydomonas sp. decreased in the order FerMEG12 > Carbo-Iron® > Fe-zeolite > Nano-Goethite. Ecotoxicological studies were challenged due to some particle properties, i.e. dark colour, effect of constituents and a tendency to agglomerate, especially at high concentrations. All particles exhibited potential to induce significant toxicity at high concentrations (500 mg L-1), though such concentrations would rapidly decrease to mg or µg L-1 in aquatic environments, levels harmless to Chlamydomonas sp. The presented findings contribute to the practical usage of particle-based nanoremediation in environmental restoration.

Magnetic Poly(N-isopropylacrylamide) Nanocomposites: Effect of Preparation Method on Antibacterial Properties.

The most challenging task in the preparation of magnetic poly(N-isopropylacrylamide) (Fe3O4-PNIPAAm) nanocomposites for bio-applications is to maximise their reactivity and stability. Emulsion polymerisation, in situ precipitation and physical addition were used to produce Fe3O4-PNIPAAm-1, Fe3O4-PNIPAAm-2 and Fe3O4-PNIPAAm-3, respectively. Their properties were characterised using scanning electron microscopy (morphology), zeta-potential (surface charge), thermogravimetric analysis (stability), vibrating sample magnetometry (magnetisation) and dynamic light scattering. Moreover, we investigated the antibacterial effect of each nanocomposite against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Both Fe3O4-PNIPAAm-1 and Fe3O4-PNIPAAm-2 nanocomposites displayed high thermal stability, zeta potential and magnetisation values, suggesting stable colloidal systems. Overall, the presence of Fe3O4-PNIPAAm nanocomposites, even at lower concentrations, caused significant damage to both E. coli and S. aureus DNA and led to a decrease in cell viability. Fe3O4-PNIPAAm-1 displayed a stronger antimicrobial effect against both bacterial strains than Fe3O4-PNIPAAm-2 and Fe3O4-PNIPAAm-3. Staphylococcus aureus was more sensitive than E. coli to all three magnetic PNIPAAm nanocomposites.

Ecotoxicity testing and environmental risk assessment of iron nanomaterials for sub-surface remediation - Recommendations from the FP7 project NanoRem.

Nanoremediation with iron (Fe) nanomaterials opens new doors for treating contaminated soil and groundwater, but is also accompanied by new potential risks as large quantities of engineered nanomaterials are introduced into the environment. In this study, we have assessed the ecotoxicity of four engineered Fe nanomaterials, specifically, Nano-Goethite, Trap-Ox Fe-zeolites, Carbo-Iron® and FerMEG12, developed within the European FP7 project NanoRem for sub-surface remediation towards a test battery consisting of eight ecotoxicity tests on bacteria (V. fisheri, E. coli), algae (P. subcapitata, Chlamydomonas sp.), crustaceans (D. magna), worms (E. fetida, L. variegatus) and plants (R. sativus, L. multiflorum). The tested materials are commercially available and include Fe oxide and nanoscale zero valent iron (nZVI), but also hybrid products with Fe loaded into a matrix. All but one material, a ball milled nZVI (FerMEG12), showed no toxicity in the test battery when tested in concentrations up to 100 mg/L, which is the cutoff for hazard labeling in chemicals regulation in Europe. However it should be noted that Fe nanomaterials proved challenging to test adequately due to their turbidity, aggregation and sedimentation behavior in aqueous media. This paper provides a number of recommendations concerning future testing of Fe nanomaterials and discusses environmental risk assessment considerations related to these.

Dual-modality self-heating and antibacterial polymer-coated nanoparticles for magnetic hyperthermia.

Multifunctional nanoparticles for magnetic hyperthermia which simultaneously display antibacterial properties promise to decrease bacterial infections co-localized with cancers. Current methods synthesize such particles by multi-step procedures, and systematic comparisons of antibacterial properties between coatings, as well as measurements of specific absorption rate (SAR) during magnetic hyperthermia are lacking. Here we report the novel simple method for synthesis of magnetic nanoparticles with shells of oleic acid (OA), polyethyleneimine (PEI) and polyethyleneimine-methyl cellulose (PEI-mC). We compare their antibacterial properties against single gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria as well as biofilms. Magnetite nanoparticles (MNPs) with PEI-methyl cellulose were found to be most effective against both S. aureus and E. coli with concentration for 10% growth inhibition (EC10) of <150 mg/l. All the particles have high SAR and are effective for heat-generation in alternating magnetic fields.

Optimized fed-batch fermentation of Scheffersomyces stipitis for efficient production of ethanol from hexoses and pentoses.

Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose-xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1 h(-1). With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7 g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.

Metabolic pathway analysis and kinetic studies for production of nattokinase in Bacillus subtilis.

We have constructed a reaction network model of Bacillus subtilis. The model was analyzed using a pathway analysis tool called elementary mode analysis (EMA). The analysis tool was used to study the network capabilities and the possible effects of altered culturing conditions on the production of a fibrinolytic enzyme, nattokinase (NK) by B. subtilis. Based on all existing metabolic pathways, the maximum theoretical yield for NK synthesis in B. subtilis under different substrates and oxygen availability was predicted and the optimal culturing condition for NK production was identified. To confirm model predictions, experiments were conducted by testing these culture conditions for their influence on NK activity. The optimal culturing conditions were then applied to batch fermentation, resulting in high NK activity. The EMA approach was also applied for engineering B. subtilis metabolism towards the most efficient pathway for NK synthesis by identifying target genes for deletion and overexpression that enable the cell to produce NK at the maximum theoretical yield. The consistency between experiments and model predictions proves the feasibility of EMA being used to rationally design culture conditions and genetic manipulations for the efficient production of desired products.

Rational optimization of culture conditions for the most efficient ethanol production in Scheffersomyces stipitis using design of experiments.

Optimization of culture parameters for achieving the most efficient ethanol fermentation is challenging due to multiple variables involved. Here we presented a rationalized methodology for multi-variables optimization through the design of experiments DoE approach. Three critical parameters, pH, temperature, and agitation speed, affecting ethanol fermentation in S. stipitis was investigated. A predictive model showed that agitation speed significantly affected ethanol synthesis. Reducing pH and temperature also improved ethanol production. The model identified the optimum culture conditions for the most efficient ethanol production with the yield and productivity of 0.46 g/g and 0.28 g/l h, respectively, which is consistent with experimental observation. The results also indicated the scalability of the model from shake flask to bioreactor. Thus, DoE is a promising tool permitting the rapid establishment of culture conditions for the most efficient ethanol fermentation in S. stipitis. The approach could be useful to reduce process development time in lignocellulosic ethanol industry.