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The performance of large-scale stirred tank and bubble column bioreactors is often hindered by insufficient macromixing of feeds, leading to heterogeneities in pH, substrate, and oxygen, which complicates process scale-up. Appropriate feed placement or the use of multiple feed points could improve mixing. Here, theoretically optimal placement of feed points was derived using one-dimensional diffusion equations. The utility of optimal multipoint feeds was evaluated with mixing, pH control, and bioreaction simulations using three-dimensional compartment models of four industrially relevant bioreactors with working volumes ranging from 8 to 237 m3. Dividing the vessel axially in equal-sized compartments and locating a feed point or multiple feed points symmetrically in each compartment reduced the mixing time substantially by more than a minute and mitigated gradients of pH, substrate, and oxygen. Performance of the large-scale bioreactors was consequently restored to ideal, homogeneous reactor performance: oxygen consumption and biomass yield were recovered and the phenotypical heterogeneity of the biomass population was diminished. publishedVersion

Bioprocesses involving more than one species can alleviate restrictions posed by limited substrate range of single species. Coupled, multistage cultures can be useful when heterogeneous substrates, such as lignocellulosic biomass, are exploited. Here, microbial production of α-olefins (C11) from lignocellulosic substrates, namely cellulose and technical lignin, was investigated. A two-stage culture with cellulose fermentation to organic acids by Clostridium cellulolyticum and subsequent upgrading of the organic acids to 1-undecene by engineered Acinetobacter baylyi ADP1 was established. As a result, A. baylyi ADP1 synthesised 107 μg L-1 of 1-undecene from cellulose. Additionally, ligninolytic effects by A. baylyi ADP1 on softwood were confirmed and downstream processing for continuous 1-undecene collection was introduced. In addition, the synthesis of poly-α-olefin trimers (C33) by the oligomerization of 1-undecene was demonstrated. This study demonstrates the potential of integrated multistage processes in treating challenging substrates. publishedVersion

Adaptive laboratory evolution (ALE) is a powerful approach for improving phenotypes of microbial hosts. Evolved strains typically contain numerous mutations that can be revealed by whole-genome sequencing. However, determining the contribution of specific mutations to new phenotypes is typically challenging and laborious. This task is complicated by factors such as the mutation type, the genomic context, and the interplay between different mutations. Here, a novel approach was developed to identify the significance of mutations in strains evolved from Acinetobacter baylyi ADP1. This method, termed rapid advantageous mutation screening and selection (RAMSES), was used to analyze mutants that emerged from stepwise adaptation to and consumption of high levels of ferulate, a common lignin-derived aromatic compound. After whole-genome sequence analysis, RAMSES allowed rapid determination of effective mutations and seamless introduction of the beneficial mutations into the chromosomes of new strains with different genetic backgrounds. This simple approach to reverse engineering exploits the natural competence and high recombination efficiency of ADP1. Mutated DNA, added directly to growing cells, replaces homologous chromosomal regions to generate transformants that will become enriched if there is a selective benefit. Thus, advantageous mutations can be rapidly identified. Here, the growth advantage of transformants under selective pressure revealed key mutations in genes related to aromatic transport, including hcaE, hcaK, and vanK, and a gene, ACIAD0482, which is associated with lipopolysaccharide synthesis. This study provided insights into the enhanced utilization of industrially relevant aromatic substrates and demonstrated the use of A. baylyi ADP1 as a convenient platform for strain development and evolution studies. IMPORTANCE Microbial conversion of lignin-enriched streams is a promising approach for lignin valorization. However, the lignin-derived aromatic compounds are toxic to cells at relevant concentrations. Although adaptive laboratory evolution (ALE) is a powerful approach to develop more tolerant strains, it is typically laborious to identify the mechanisms underlying phenotypic improvement. We employed Acinetobacter baylyi ADP1, an aromatic-compound-degrading strain that may be useful for biotechnology. The natural competence and high recombination efficiency of this strain can be exploited for critical applications, such as the breakdown of lignin and plastics and abundant polymers composed of aromatic subunits. The natural transformability of this bacterium enabled us to develop a novel approach for rapid screening of advantageous mutations from ALE-derived, aromatic-tolerant, ADP1-derived strains. We clarified the mechanisms and genetic targets for improved tolerance toward common lignin-derived aromatic compounds. This study facilitates metabolic engineering for lignin valorization.

Engineered microbial consortia can provide several advantages over monocultures in terms of utilization of mixed substrates, resistance to perturbations, and division of labor in complex tasks. However, maintaining stability, reproducibility, and control over population levels in variable conditions can be challenging in multispecies cultures. In our study, we modeled and constructed a synthetic symbiotic consortium with a genetically encoded carbon cross-feeding system. The system is based on strains of Escherichia coli and Acinetobacter baylyi ADP1, both engineered to be incapable of growing on glucose on their own. In a culture supplemented with glucose as the sole carbon source, growth of the two strains is afforded by the exchange of gluconate and acetate, resulting in inherent control over carbon availability and population balance. We investigated the system robustness in terms of stability and population control under different inoculation ratios, substrate concentrations, and cultivation scales, both experimentally and by modeling. To illustrate how the system might facilitate division of genetic circuits among synthetic microbial consortia, a green fluorescent protein sensitive to pH and a slowly maturing red fluorescent protein were expressed in the consortium as measures of a circuit's susceptibility to external and internal variability, respectively. The symbiotic consortium maintained stable and linear growth and circuit performance regardless of the initial substrate concentration or inoculation ratio. The developed cross-feeding system provides simple and reliable means for population control without expression of non-native elements or external inducer addition, being potentially exploitable in consortia applications involving precisely defined cell tasks or division of labor. acceptedVersion

Crude glycerol is an excellent carbon source for bacterial production systems. Bacterial fermentation often generates by-products that can offer an additional carbon pool to improve the product profile for optimal valorization. In this study, the properties of two phylogenetically distinct bacteria, Acinetobacter baylyi ADP1 and Clostridium butyricum, were coupled in a one-pot batch process to co-produce 1,3 propanediol (PDO) and long-chain alkyl esters (wax esters, WEs) from crude glycerol. In the process, A. baylyi deoxidized the growth medium allowing glycerol fermentation and PDO production by C. butyricum. Reaeration of the co-cultivations enabled A. baylyi to metabolize the fermentation by-products, acetate and butyrate, and synthesize intracellular WEs. To improve PDO production and A. baylyi growth, carbon and macronutrients in the growth medium were screened and optimized using Plackett-Burman and Box-Behnken models. The validation experiment revealed a good correlation between the observed and predicted values. The salting-out method recovered 89.5% PDO from the fermentation broth and in vacuo extraction resulted in a PDO content of 5.3 g L-1. Nuclear magnetic resonance revealed a WE content and yield of 34.4 ± 1.4 mg L-1 and 34.2 ± 3.2 mg WE g-1 dry cell weight, respectively. A molar yield of 0.65 mol PDO mol-1 and 0.62 µmol WE mol-1 crude glycerol was achieved with the synthetic consortium. This work emphasizes the strength of response surface methodology in improving production processes from the mutualistic association of divergent bacterial species in consortium. The co-production of PDO and WEs from crude glycerol is demonstrated for the first time in this study. acceptedVersion

Plastic debris degrades in the water environments due to various factors such as mechanical stress. Small-sized degradation products, including plastic monomers, are currently monitored using equipment which might be unsuitable for screening. Here, we developed a recombinant whole-cell bacterial biosensor, which could be used for this type of monitoring. The Escherichia coli pBAV1K-ACU-lucFF cells contain a luciferase-based reporter system under the control of acrylic acid specific promoter. The biosensor cells were used to detect acrylic acid monomers from both sterile water and spiked lake water samples, indicating usability with environmental samples. Furthermore, poly(acrylic acid) was incubated in salt water, and the biosensor cells could identify acrylic acid monomers originating from it. Thus, the cells could be used to observe similar processes in the environment. The results show that the bacterial biosensors could complement the current research methods of plastic monomer monitoring in water environments with a potential for higher throughputs. publishedVersion

Adaptive laboratory evolution (ALE) is a powerful approach for improving phenotypes of microbial hosts. Evolved strains typically contain numerous mutations that can be revealed by whole-genome sequencing. However, determining the contribution of specific mutations to new phenotypes is typically challenging and laborious. This task is complicated by factors such as the mutation type, the genomic context, and the interplay between different mutations. Here, a novel approach was developed to identify the significance of mutations in strains derived from Acinetobacter baylyi ADP1. This method, termed Rapid Advantageous Mutation ScrEening and Selection (RAMSES), was used to analyze mutants that emerged from stepwise adaptation to, and consumption of, high levels of ferulate, a common lignin-derived aromatic compound. After whole-genome sequence analysis, RAMSES allowed both rapid determination of effective mutations and seamless introduction of the beneficial mutations into the chromosomes of new strains with different genetic backgrounds. This simple approach to reverse-engineering exploits the natural competence and high recombination efficiency of ADP1. The growth advantage of transformants under selective pressure revealed key mutations in genes related to aromatic transport, including hcaE, hcaK, and vanK, and a gene, ACIAD0482, which is associated with lipopolysaccharide synthesis. This study provides insights into enhanced utilization of industrially relevant aromatic substrates and demonstrates the use of A. baylyi ADP1 as a convenient platform for strain development and evolution studies.ImportanceMicrobial conversion of lignin-enriched streams is a promising approach for lignin valorization. However, the lignin-derived aromatic compounds are toxic to cells at relevant concentrations. Adaptive laboratory evolution is a powerful approach to develop more tolerant strains, but revealing the underlying mechanisms behind phenotypic improvement typically involves laborious processes. We employed Acinetobacter baylyi ADP1, an aromatic compound degrading strain that may be useful for biotechnology. The natural competence and high recombination efficiency of strain ADP1 can be exploited for critical applications such as the breakdown of lignin and plastics, abundant polymers composed of aromatic subunits. The natural transformability of this bacterium enabled us to develop a novel approach that allows rapid screening of advantageous mutations from ALE-derived aromatic-tolerant ADP1 strains. We clarified the mechanisms and genetic targets for improved tolerance towards common lignin-derived aromatic compounds. This study facilitates metabolic engineering for lignin valorization.

Methane (CH4) is a sustainable carbon feedstock for value-added chemical production in aerobic CH4-oxidizing bacteria (methanotrophs). Under substrate-limited (e.g., oxygen and nitrogen) conditions, CH4 oxidation results in the production of various short-chain organic acids and platform chemicals. These CH4-derived products could be broadened by utilizing them as feedstocks for heterotrophic bacteria. As a proof of concept, a two-stage system for CH4 abatement and 1-alkene production was developed in this study. Type I and Type II methanotrophs, Methylobacter tundripaludum SV96 and Methylocystis rosea SV97, respectively, were investigated in batch tests under different CH4 and air supplementation schemes. CH4 oxidation under either microaerobic or aerobic conditions induced the production of formate, acetate, succinate, and malate in M. tundripaludum SV96, accounting for 4.8–7.0% of consumed carbon from CH4 (C-CH4), while M. rosea SV97 produced the same compounds except for malate, and with lower efficiency than M. tundripaludum SV96, accounting for 0.7–1.8% of consumed C-CH4. For the first time, this study demonstrated the use of organic acid-rich spent media of methanotrophs cultivating engineered Acinetobacter baylyi ADP1 ‘tesA-undA cells for 1-alkene production. The highest yield of 1-undecene was obtained from the spent medium of M. tundripaludum SV96 at 68.9 ± 11.6 μmol mol Csubstrate–1. However, further large-scale studies on fermenters and their optimization are required to increase the production yields of organic acids in methanotrophs.

Komagataeibacter spp. has been used for the bioconversion of industrial wastes and lignocellulosic hydrolysates to bacterial cellulose (BC). Recently, studies have demonstrated the capacity of Komagataeibacter spp. in the biotransformation of inhibitors found in lignocellulosic hydrolysates, aromatic lignin-derived monomers (LDMs) and acetate. In general, detoxification and BC synthesis from lignocellulosic inhibitors requires a carbon flow from acetyl-coA towards tricarboxylic acid and gluconeogenesis, respectively. However, the related molecular aspects have not yet been identified in Komagataeibacter spp. In this study, we isolated a cellulose-producing bacterium capable of synthesizing BC in a minimal medium containing crude glycerol, a by-product from the biodiesel production process. The isolate, affiliated to Komagataeibacter genus, synthesized cellulose in a minimal medium containing glucose (3.3 ± 0.3 g/L), pure glycerol (2.2 ± 0.1 g/L) and crude glycerol (2.1 ± 0.1 g/L). Genome assembly and annotation identified four copies of bacterial cellulose synthase operon and genes for redirecting the carbon from the central metabolic pathway to gluconeogenesis. According to the genome annotations, a BC production route from acetyl-CoA, a central metabolic intermediate, was hypothesized and was validated using acetate. We identified that when K. rhaeticus ENS9b was grown in a minimal medium supplemented with acetate, BC production was not observed. However, in the presence of readily utilizable substrates, such as spent yeast hydrolysate, acetate supplementation improved BC synthesis.

Komagataeibacter spp. has been used for the bioconversion of industrial wastes and lignocellulosic hydrolysates to bacterial cellulose (BC). Recently, studies have demonstrated the capacity of Komagataeibacter spp. in the biotransformation of inhibitors found in lignocellulosic hydrolysates, aromatic lignin-derived monomers (LDMs) and acetate. In general, detoxification and BC synthesis from lignocellulosic inhibitors requires a carbon flow from acetyl-coA towards tricarboxylic acid and gluconeogenesis, respectively. However, the related molecular aspects have not yet been identified in Komagataeibacter spp. In this study, we isolated a cellulose-producing bacterium capable of synthesizing BC in a minimal medium containing crude glycerol, a by-product from the biodiesel production process. The isolate, affiliated to Komagataeibacter genus, synthesized cellulose in a minimal medium containing glucose (3.3 ± 0.3 g/L), pure glycerol (2.2 ± 0.1 g/L) and crude glycerol (2.1 ± 0.1 g/L). Genome assembly and annotation identified four copies of bacterial cellulose synthase operon and genes for redirecting the carbon from the central metabolic pathway to gluconeogenesis. According to the genome annotations, a BC production route from acetyl-CoA, a central metabolic intermediate, was hypothesized and was validated using acetate. We identified that when K. rhaeticus ENS9b was grown in a minimal medium supplemented with acetate, BC production was not observed. However, in the presence of readily utilizable substrates, such as spent yeast hydrolysate, acetate supplementation improved BC synthesis.

Microbial storage compounds, such as wax esters (WE), are potential high-value lipids for the production of specialty chemicals and medicines. Their synthesis, however, is strictly regulated and competes with cell growth, which leads to trade-offs between biomass and product formation. Here, we use metabolic engineering and synergistic substrate cofeeding to partition the metabolism of Acinetobacter baylyi ADP1 into two distinct modules, each dedicated to cell growth and WE biosynthesis, respectively. We first blocked the glyoxylate shunt and upregulated the WE synthesis pathway to direct the acetate substrate exclusively for WE synthesis, then we controlled the supply of gluconate so it could be used exclusively for cell growth and maintenance. We show that the two modules are functionally independent from each other, allowing efficient lipid accumulation while maintaining active cell growth. Our strategy resulted in 7.2- and 4.2-fold improvements in WE content and productivity, respectively, and the product titer was enhanced by 8.3-fold over the wild type strain. Notably, during a 24-hour cultivation, a yield of 18% C-WE/C-total-substrates was achieved, being the highest reported for WE biosynthesis. This work provides a simple, yet powerful, means of controlling cellular operations and overcoming some of the fundamental challenges in microbial storage lipid production. This article is protected by copyright. All rights reserved.

Tall vessels equipped with multiple impellers, such as many gas-liquid contactors in bioprocessing, often suffer from poor macromixing of feeds, which leads to heterogeneities and complicates the scale-up. Here, a diffusion equation was used as a simple model of macromixing in multi-impeller tanks, and the model fitted previously published experimental tracer curves. The model was then noted to predict that the macromixing time-scale is decreased to a quarter by relocating the feed point from the top to the middle. Furthermore, dividing the vessel axially in symmetric proportions and locating a feed point in the center of each compartment was found to reduce the time-scale even further. These theoretical results were put to test by compartment model simulations of 17 L to 22 m3 tanks with one to four impellers. Order-of-magnitude improvements were seen in the multi-impeller setups: up to 29-fold macromixing rates compared to a single-point top feed were realized with multiple feed points in a 22 m3 bioreactor stirred with four Rushton turbines. Considerable improvements were observed also in the other scales and geometries with fewer than four impellers, which suggests that the herein proposed division of feed points may greatly facilitate the scale-up of tall multi-impeller vessels.

Lignin has potential as a sustainable feedstock for microbial production of industrially relevant molecules. However, the required lignin depolymerization yields a heterogenic mixture of aromatic monomers that are challenging substrates for the microorganisms commonly used in the industry. Here, we investigated the properties of lignin-related aromatic compounds (LRAs), namely coumarate, ferulate, and caffeate, in the synthesis of biomass and products in an LRA-utilizing bacterial host Acinetobacter baylyi ADP1. The biosynthesis products, wax esters, and alkanes are relevant compounds for the chemical and fuel industries. Here, wax esters were produced by a native pathway of ADP1, whereas alkanes were produced by a synthetic pathway introduced to the host. Using individual LRAs as substrates, the growth and product formation were monitored with internal biosensors and off-line analytics. Of the tested LRAs, coumarate was the most propitious in terms of product synthesis. Wax esters were produced from coumarate with yield and titer of 37 mg/gcoumarate and 202 mg/L, whereas alkanes were produced with a yield of 62.3 µg /gcoumarate and titer of 152 µg/L. This study demonstrates the microbial preference for certain LRAs and highlights the potential of A. baylyi ADP1 as a host for LRA upgrading to value-added products.

Many genetic reporter systems require molecular oxygen; therefore, the use of reporter genes to study molecular mechanisms in anaerobic microorganisms has been hampered by the lack of convenient reporting systems. We describe reporter gene whole cell-based biosensor systems based on luciferase genes and the associated oxygen-requiring enzymes. By using two different oxygen-dependent reporters, insect and bacterial luciferases, and two bacterial hosts, Gram (+) Bifidobacterium longum and Gram (-) Escherichia coli, we show that the enzymes can be used in gene expression studies of anaerobic bacteria. E. coli, a facultative anaerobe, was grown both in aerobic and anaerobic conditions with an arabinose-inducible expression system. We show that a short treatment time of few minutes in ambient atmosphere is sufficient to detect light emission from living cells that is directly proportional to the number of cells and to the inducer concentration. The induction levels were the same in both the aerobically and anaerobically cultured cells. Similar results were obtained in the case of B. longum cultured in anaerobic conditions. publishedVersion

Acinetobacter baylyi ADP1 is a non-pathogenic soil bacterium known for its metabolic diversity and high natural transformation and recombination efficiency. For these features, A. baylyi ADP1 has been long exploited in studying bacterial genetics and metabolism. The large pool of information generated in the fundamental studies has facilitated the development of a broad range of sophisticated and robust tools for the genome and metabolic engineering of ADP1. This mini-review outlines and describes the recent advances in ADP1 engineering and tool development, exploited in, for example, pathway and enzyme evolution, genome reduction and stabilization, and for the production of native and non-native products in both pure and rationally designed multispecies cultures. The rapidly expanding toolbox together with the unique features of A. baylyi ADP1 provide a strong base for a microbial cell factory excelling in synthetic biology applications where evolution meets rational engineering.

Abstract Bacterial cellulose (BC) is a biodegradable polymer that benefits in purity, crystallinity and superior optical, structural and mechanical properties. Such properties facilitate BC to replace the conventional non-biodegradable materials used, for instance, in sensing applications. However, BC production is largely conducted in conventional medium containing model substrates and complex carbon-containing compounds. Aiming towards the production of eco-friendly piezoelectric-responsive BC films, we isolated and characterized a novel bacterial strain affiliated to Komagataeibacter rhaeticus. The K. rhaeticus ENS9a strain synthesized BC in minimal medium containing crude glycerol, generating a titer of 2.9 ± 0.3 g/L BC. This is, to the best of our knowledge, the highest BC titer reported from an unoptimized minimal medium containing crude glycerol. Interestingly, the films prepared from crude glycerol showed normal force and bending mode sensitivities of 6–11 pC/N and 40–71 pC/N, respectively, demonstrating a green platform to address both bioprocess waste valorization and implementation of cellulose-based alternatives for the non-sustainable and non-biodegradable materials, such as fluoropolymers or lead containing piezoceramics, used in sensing applications. In silico genome analysis predicted genes partaking in carbohydrate metabolism, BC biogenesis, and nitrogen fixation/regulation. Graphic abstract

Whole-cell bacterial biosensors can be applied for the screening of antibacterial properties of extracts. We constructed a biosensor panel consisting of four different bacterial biosensor strains: Escherichia, Staphylococcus, Acinetobacter and Pseudomonas for expanded screening potential. The functionality of the panel was first evaluated with known antibacterial compounds: ethanol, naphthoquinones (juglone, lawsone, plumbagin) and a flavonoid (quercetin). Natural extracts comprise a vast source of potential new antibacterials for diverse functional purposes. To demonstrate the utilization of the panel for screening of a demanding sample material, round-leaved sundew (Drosera rotundifolia) extracts were used as an example. Differences between field- and laboratory originating sundew extracts could be detected. This demonstrates the efficiency of the developed biosensor panel in the rapid screening of the antibacterial properties of plant extracts. acceptedVersion

Despite the continuing interest in various plant and natural products, only a small portion of the biologically active compounds from nature has been discovered and exploited. In this study, antioxidant and antibacterial properties of aqueous fractions of three endophytic fungi isolated from the roots of 8-year-old Scots pines (Pinus sylvestris) growing on a drained peatland were investigated. The endophytic fungi species were Acephala applanata, Phialocephala fortinii, and Humicolopsis cephalosporioides/Coniochaeta mutabilis. The bioactivities were examined using hydrogen peroxide scavenging and oxygen radical absorbance capacity tests as well as sensitive Escherichia coli-based biosensors, which produce a luminescent signal in the presence of substances with oxidative or genotoxic properties. In addition, cell models for Parkinsonʼs disease, age-related macular degeneration, and osteoarthritis were used to evaluate the potential for pharmaceutical applications. The aqueous extracts of fungi and 19 out of 42 fractions were found to be active in one or more of the tests used. However, no activity was found in the age-related macular degeneration and osteoarthritis cell model tests. Additionally, bioactivity data was connected with metabolites putatively annotated, and out of 330 metabolites, 177 were interesting in view of the bioactivities investigated. A majority of these were peptides and all three fungal species shared a highly similar metabolome. We propose that Scots pine endophytic fungi are a rich source of interesting metabolites, and synergistic effects may cause the bioactivities, as they were found to vary after the fractionation process.

Low-cost and highly sensitive piezoelectric sensors were fabricated from bacterial cellulose (BC)/MnFe2O4 nanocomposite films via a co-precipitation method, followed by hot-pressing. MnFe2O4 nanoparticles were homogeneously distributed in the BC structure. The piezoelectric sensitivity measurements in the normal mode showed that the pristine BC film exhibited a sensitivity of ∼5 pC/N, whereas this value was increased to 23 pC/N for the composite film, which is comparable to the PVDF reference film. In the bending mode, the piezoelectric response increased to 25 pC/N and 57 pC/N for the BC film and the composite film, respectively. Moreover, the piezoelectric sensitivity was significantly enhanced using carbon tape electrodes attached directly to the films instead of sandwiched electrodes. This produced a sensitivity of greater than 50 pC/N for the MBC nanocomposite film in the normal mode measurement. Our work demonstrates the potential of using MBC composite films as inexpensive and highly sensitive flexible piezoelectric sensors. acceptedVersion

Social media makes available vast amounts of data for various types of analyses. Cities have the opportunity to explore this new data source to study urban dynamics and complement traditional data used for urban planning. We investigate Untappd social media data in the context of urban planning in Curitiba, Brazil. We analyze the project to create a Craft Beer Street, recently announced by the municipality to promote local beers in Curitiba, in order to study the potential of exploring social media data to support the planning of this project. Our results indicate that social media data could have helped to guide the decision of the Beer Street creation and can potentially become a strategic urban planning tool.

The properties and antimicrobial efficacies of zinc complexes of tetrakis(N-methylpyridinium-4-yl) tetraiodide porphyrin and tetrakis(N-methylpyridinium-4-yl) tetraiodide phthalocyanine impregnated to paper were evaluated. Photo-inactivation of microbes using inexpensive consumer light-emitting diode lamp was assessed on surface of dyed papers. Antimicrobial experiments of phthalocyanine-dyed paper by live cell assessment through colony forming units counting demonstrated 3.72 and 4.01 log reduction against Escherichia coli (E. coli) and Acinetobacter baylyi (A. baylyi) respectively after 1 h of illumination with 35 mW/cm2 light. The porphyrin-dyed paper exhibited 1.66 and 2.01 log reduction in colony forming units against E. coli and A. baylyi respectively after 1 h exposure with 4 mW/cm2 light. Both dyed papers were photo-stable after 64 h of continuous exposure with 42 mW/cm2 light, while phthalocyanine-dyed paper exhibited superior leaching stability in phosphate-buffered saline.

The pulp and paper industry together with lignocellulosic biofuel production provides plentiful streams of lignin and lignin-derived molecules (LDMs) that currently remain underutilized. The heterogeneity and complexity of lignin along with the lack of convenient tools significantly hamper its utilization. Selective separation of these LDMs from streams using specific tools would allow the recovery of aromatic compounds, as well as facilitate biological processes aiming at lignin valorization. To this end, here we report the isolation and characterization of single-chain variable fragment (scFv) antibodies against ferulate, coumarate, and caffeate, which are the molecular representatives of LDMs. Binders for the target LDMs were enriched by interrogating a synthetic scFv library with the phage display technique. As a result, scFv binders specific against each of the target molecules were obtained with affinities in the micromolar range. The selectivity of scFvs towards specific LDMs was proved by recovering caffeate from simulated LDM solution, Kraft lignin, and rice straw hydrolysate samples. Further proof of concept studies with model compounds demonstrated the applicability of antibody-based binders as a detection tool for monitoring microbial LDM conversion. Overall, this study demonstrates the potential of scFv binders as a specific toolset for lignin compound recovery and analysis. publishedVersion

We have synthesized novel phthalocyanine with four pyridyl substituents connected to α-phthalo-positions via direct C-C bond. The Zn complex and tetracationic derivatives of phthalocyanine were also synthesized and the dyes were impregnated into filter paper to prepare photoactive antimicrobial surface. The photodynamic antimicrobial efficacy of the dyed paper samples was evaluated by a simple and fast setup using bioluminescent microbes. Escherichia coli and Acinetobacter baylyi ADP1 strains carrying bacterial luciferase genes were used in the screening experiment. The most efficient compound, tetracationic zinc derivative 8, was investigated further. The compound was highly water soluble, had high molar absorptivity and exhibited good adhesion to the filter paper without leaching into the solution. The singlet oxygen quantum yield of tetracationic zinc derivative 8 in water was found out to be 30 ± 20%. According to the cell viability assay test performed on E. coli wild type in solution, the molecule had similar or better photo toxicity as the reference photosensitizer, tetrakis (1-methyl-pyridinium-4-yl)porphyrin (TMPyP). Antimicrobial efficacy of the dye 8 on photoactive surface was studied by live cell assessment through colony forming unit (CFU) counting. The colored surface demonstrated 3 log reduction in CFU against E. coli and A. baylyi ADP1 just after 1 h of illumination with the white light of low intensity.

Low-cost and highly sensitive piezoelectric sensors were fabricated from bacterial cellulose (BC)/MnFe

Endophytes are microorganisms living inside plant hosts and are known to be beneficial for the host plant vitality. In this study, we isolated three endophytic fungus species from the roots of Scots pine seedlings growing on Finnish drained peatland setting. The isolated fungi belonged to dark septate endophytes (DSE). The metabolic profiles of the hot water extracts of the fungi were investigated using Ultrahigh Performance Liquid Chromatography with Diode Array Detection and Electron Spray Ionization source Mass Spectrometry with Orbitrap analyzer (UPLC–DAD–ESI–MS–Orbitrap). Out of 318 metabolites, we were able to identify 220, of which a majority was amino acids and peptides. Additionally, opine amino acids, amino acid quinones, Amadori compounds, cholines, nucleobases, nucleosides, nucleotides, siderophores, sugars, sugar alcohols and disaccharides were found, as well as other previously reported metabolites from plants or endophytes. Some differences of the metabolic profiles, regarding the amount and identity of the found metabolites, were observed even though the fungi were isolated from the same host. Many of the discovered metabolites have been described possessing biological activities and properties, which may make a favorable contribution to the host plant nutrient availability or abiotic and biotic stress tolerance.

Metabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal. An example of a synthesis pathway strongly dependent on external conditions is the production of storage lipids, which typically requires high carbon/nitrogen ratio.Acinetobacter baylyiADP1 is known for its ability to produce industrially interesting storage lipids, namely wax esters (WEs). Here, we engineered the central carbon metabolism ofA. baylyiADP1 by deletion of the geneaceAencoding for isocitrate lyase in order to allow redirection of carbon towards WEs. The production was further enhanced by overexpression of fatty acyl-CoA reductase Acr1 in the wax ester production pathway. This strategy led to 3-fold improvement in yield (0.075 g/g glucose) and 3.15-fold improvement in titer (1.82 g/L) and productivity (0.038 g/L/h) by a simple one-stage batch cultivation with glucose as carbon source. The engineered strain accumulated up to 27% WEs of cell dry weight. The titer and cellular WE content are the highest reported to date among microbes. We further showed that the engineering strategy alleviated the inherent requirement for high carbon/nitrogen ratio and demonstrated the production of wax esters using nitrogen-rich substrates including casamino acids, yeast extract and baker’s yeast hydrolysate, which support biomass production but not WE production in wild-type cells. The study demonstrates the power of metabolic engineering in overcoming natural limitations in the production of storage lipids.

Metabolic engineering can be used as a powerful tool to redirect cell resources towards product synthesis, also in conditions that are not optimal for the production. An example of synthesis strongly dependent on external conditions is the production of storage lipids, which typically requires a high carbon/nitrogen ratio. This requirement also limits the use of abundant nitrogen-rich materials, such as industrial protein by-products, as substrates for lipid production. Acinetobacter baylyi ADP1 is known for its ability to produce industrially interesting storage lipids, namely wax esters (WEs). Here, we engineered A. baylyi ADP1 by deleting the gene aceA encoding for isocitrate lyase and overexpressing fatty acyl-CoA reductase Acr1 in the wax ester production pathway to allow redirection of carbon towards WEs. This strategy led to 3-fold improvement in yield (0.075 ​g/g glucose) and 3.15-fold improvement in titer (1.82 ​g/L) and productivity (0.038 ​g/L/h) by a simple one-stage batch cultivation with glucose as carbon source. The engineered strain accumulated up to 27% WEs of cell dry weight. The titer and cellular WE content are the highest reported to date among microbes. We further showed that the engineering strategy alleviated the inherent requirement for high carbon/nitrogen ratio and demonstrated the production of wax esters using nitrogen-rich substrates including casamino acids, yeast extract, and baker’s yeast hydrolysate, which support biomass production but not WE production in wild-type cells. The study demonstrates the power of metabolic engineering in overcoming natural limitations in the production of storage lipids., Highlights • Efficient wax ester (WE) accumulation with low carbon/nitrogen ratio was demonstrated • Deletion of aceA and overexpression of acr1 enhanced WE production in Acinetobacter baylyi ADP1 • The highest WE titer, productivity, and cellular WE content reported to date was achieved • Engineered strain produced WEs directly from nitrogen-rich substrates • Metabolic engineering alleviated the native restrictions behind WE synthesis

Engineered microbial consortia can provide several advantages over monocultures in terms of utilization of mixed substrates, resistance to perturbations, and division of labor in complex tasks. However, maintaining stability, reproducibility, and control over population levels in variable conditions can be challenging in multi-species cultures. In our study, we modeled and constructed a synthetic symbiotic consortium with a genetically encoded carbon cross-feeding system. The system is based on strains ofEscherichia coliandAcinetobacter baylyiADP1, both engineered to be incapable of growing on glucose on their own. In a culture supplemented with glucose as the sole carbon source, growth of the two strains is afforded by the exchange of gluconate and acetate, resulting in inherent control over carbon availability and population balance. We investigated the system robustness in terms of stability and population control under different inoculum ratios, substrate concentrations, and cultivation scales, both experimentally and by modeling. To illustrate how the system might facilitate division of genetic circuits among synthetic microbial consortia, a green fluorescent protein sensitive to pH and a slowly-maturing red fluorescent protein were expressed in the consortium as measures of a circuit’s susceptibility to external and internal variability, respectively. The symbiotic consortium maintained stable and linear growth and circuit performance regardless of the initial substrate concentration or inoculum ratios. The developed cross-feeding system provides simple and reliable means for population control without expression of non-native elements or external inducer addition, being potentially exploitable in consortia applications involving precisely defined cell tasks or division of labor.

Endophytes are microorganisms living inside plant hosts and are known to be beneficial for the host plant vitality. In this study, we isolated three endophytic fungus species from the roots of Scots pine seedlings growing on Finnish drained peatland setting. The isolated fungi belonged to dark septate endophytes (DSE). The metabolic profiles of the hot water extracts of the fungi were investigated using Ultrahigh Performance Liquid Chromatography with Diode Array Detection and Electron Spray Ionization source Mass Spectrometry with Orbitrap analyzer (UPLC&ndash, DAD&ndash, ESI&ndash, MS&ndash, Orbitrap). Out of 318 metabolites, we were able to identify 220, of which a majority was amino acids and peptides. Additionally, opine amino acids, amino acid quinones, Amadori compounds, cholines, nucleobases, nucleosides, nucleotides, siderophores, sugars, sugar alcohols and disaccharides were found, as well as other previously reported metabolites from plants or endophytes. Some differences of the metabolic profiles, regarding the amount and identity of the found metabolites, were observed even though the fungi were isolated from the same host. Many of the discovered metabolites have been described possessing biological activities and properties, which may make a favorable contribution to the host plant nutrient availability or abiotic and biotic stress tolerance.

For improving the microbial production of fuels and chemicals, gene knock-outs and overexpression are routinely applied to intensify the carbon flow from substrate to product. However, their possibilities in dynamic control of the flux between the biomass and product synthesis are limited, whereas dynamic metabolic switches can be used for optimizing the distribution of carbon and resources. The production of single cell oils is especially challenging, as the synthesis is strictly regulated, competes directly with biomass, and requires defined conditions, such as nitrogen limitation. Here, we engineered a metabolic switch for redirecting carbon flow from biomass to wax ester production in Acinetobacter baylyi ADP1 using acetate as a carbon source. Isocitrate lyase, an essential enzyme for growth on acetate, was expressed under an arabinose inducible promoter. The autonomous downregulation of the expression is based on the gradual oxidation of the arabinose inducer by a glucose dehydrogenase gcd. The depletion of the inducer, occurring simultaneously to acetate consumption, switches the cells from a biomass mode to a lipid synthesis mode, enabling the efficient channelling of carbon to wax esters in a simple batch culture. In the engineered strain, the yield and titer of wax esters were improved by 3.8 and 3.1 folds, respectively, over the control strain. In addition, the engineered strain accumulated wax esters 19% of cell dry weight, being the highest reported among microbes. The study provides important insights into the dynamic engineering of the biomass-dependent synthesis pathways for the improved production of biocompounds from low-cost and sustainable substrates. Keywords: Lipid biosynthesis, Wax esters, Acetate, Dynamic control, Decoupling, Autonomous circuit

Background Integration of synthetic metabolic pathways to catabolically diverse chassis provides new opportunities for sustainable production. One attractive scenario is the use of abundant waste material to produce a readily collectable product, which can reduce the production costs. Towards that end, we established a cellular platform for the production of semivolatile medium-chain α-olefins from lignin-derived molecules: we constructed 1-undecene synthesis pathway in Acinetobacter baylyi ADP1 using ferulate, a lignin-derived model compound, as the sole carbon source for both cell growth and product synthesis. Results In order to overcome the toxicity of ferulate, we first applied adaptive laboratory evolution to A. baylyi ADP1, resulting in a highly ferulate-tolerant strain. The adapted strain exhibited robust growth in 100 mM ferulate while the growth of the wild type strain was completely inhibited. Next, we expressed two heterologous enzymes in the wild type strain to confer 1-undecene production from glucose: a fatty acid decarboxylase UndA from Pseudomonas putida, and a thioesterase ‘TesA from Escherichia coli. Finally, we constructed the 1-undecene synthesis pathway in the ferulate-tolerant strain. The engineered cells were able to produce biomass and 1-undecene solely from ferulate, and excreted the product directly to the culture headspace. Conclusions In this study, we employed a bacterium Acinetobacter baylyi ADP1 to integrate a natural aromatics degrading pathway to a synthetic production route, allowing the upgradation of lignin derived molecules to value-added products. We developed a highly ferulate-tolerant strain and established the biosynthesis of an industrially relevant chemical, 1-undecene, solely from the lignin-derived model compound. This study reports the production of alkenes from lignin derived molecules for the first time and demonstrates the potential of lignin as a sustainable resource in the bio-based synthesis of valuable products. Electronic supplementary material The online version of this article (10.1186/s12934-019-1097-x) contains supplementary material, which is available to authorized users.

BackgroundMicrobial biosynthesis of alkanes is considered a promising method for the sustainable production of drop-in fuels and chemicals. Carbon dioxide would be an ideal carbon source for these production systems, but efficient production of long carbon chains from CO2is difficult to achieve in a single organism. A potential solution is to employ acetogenic bacteria for the reduction of CO2to acetate, and engineer a second organism to convert the acetate into long-chain hydrocarbons.ResultsIn this study, we demonstrate alkane production from CO2by a system combining the acetogenAcetobacterium woodiiand a non-native alkane producerAcinetobacter baylyiADP1 engineered for alkane production. Nine synthetic two-step alkane biosynthesis pathways consisting of different aldehyde- and alkane-producing enzymes were combinatorically constructed and expressed inA. baylyi.The aldehyde-producing enzymes studied were AAR fromSynechococcus elongatus,Acr1 fromA. baylyi,and Ramo, a putative dehydrogenase, fromNevskia ramosa.The alkane-producing enzymes were ADOs fromS. elongatusandNostoc punctiforme,and CER1 fromArabidopsis thaliana.The performance of the pathways was evaluated with a twin-layer biosensor, which allowed the monitoring of both the intermediate, fatty aldehyde, as well as the alkane production. The highest alkane production, as indicated by the biosensor, was achieved with a pathway consisting of AAR and ADO fromS. elongatus.The performance of this pathway was further improved by balancing the relative expression levels of the enzymes in order to limit the accumulation of the intermediate fatty aldehyde. Finally, the acetogenA. woodiiwas used to produce acetate from CO2and H2, and the acetate was used for alkane production by the engineeredA. baylyi,thereby leading to the net production of long-chain alkanes from CO2.ConclusionsA modular system for the production of drop-in liquid fuels from CO2was demonstrated. Among the studied synthetic pathways, the combination of ADO and AAR fromS. elongatuswas found to be the most efficient in heterologous alkane production inA. baylyi.Furthermore, limiting the accumulation of the fatty aldehyde intermediate was found to be beneficial for the alkane production.

The authors wish to make the following corrections to this paper published in Molecules[...]

Many genetic reporter systems require molecular oxygen; therefore, the use of reporter genes to study molecular mechanisms in anaerobic microorganisms has been hampered by the lack of convenient reporting systems. We describe reporter gene whole cell-based biosensor systems based on luciferase genes and the associated oxygen-requiring enzymes. By using two different oxygen-dependent reporters, insect and bacterial luciferases, and two bacterial hosts, Gram (+) Bifidobacterium longum and Gram (-) Escherichia coli, we show that the enzymes can be used in gene expression studies of anaerobic bacteria. E. coli, a facultative anaerobe, was grown both in aerobic and anaerobic conditions with an arabinose-inducible expression system. We show that a short treatment time of few minutes in ambient atmosphere is sufficient to detect light emission from living cells that is directly proportional to the number of cells and to the inducer concentration. The induction levels were the same in both the aerobically and anaerobically cultured cells. Similar results were obtained in the case of B. longum cultured in anaerobic conditions.

Ultraviolet radiation (UV-R) causes genotoxic and aging effects on skin, and sunscreens are used to alleviate the damage. However, sunscreens contain synthetic shielding agents that can cause harmful effects in the environment. Nature-derived substances may have potential as replacement materials for the harmful sunscreen chemicals. However, screening of a broad range of samples is tedious, and often requires a separate genotoxicity assessment. We describe a simple microplate technique for the screening of UV protective substances using a recombinant Escherichia coli biosensor. Both absorbance-based and bioactivity-based shields can be detected with simultaneous information about the sample genotoxicity. With this technique, a controversial sunscreen compound, oxybenzone offers physical or absorbance-based shield but appears genotoxic at higher concentrations (3.3 mg/mL). We also demonstrate that pine needle extract (PiNe ) shields the biosensor from UV-R in a dose-dependent manner without showing genotoxicity. The physical shield of 5 mg/mL PiNe was similar to that of one of the most common UV-shielding compound TiO2 concentration 0.80 mg/mL. The bioactivity-based shield of PiNe also reaches the extent of the physical shield with the highest concentration (3.3 mg/mL). We conclude that our technique is suitable in detecting the UV-shielding potential of natural substances, and gives simultaneous information on genotoxicity.