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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

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.

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

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.

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

This study demonstrates the potential for biohydrogen production in a co-culture of two ecologically distant species, Thermatoga neapolitana and Caldicellulosiruptor saccharolyticus, and the development of a quantitative real-time PCR (qPCR) method for quantifying the hyperthermophilic bacterium of the genus Thermotoga. Substrate utilization and H2 production performance was compared to those of their individual cultures. The highest H2 yields obtained were 2.7 ± 0.05, 2.5 ± 0.07 and 2.8 ± 0.09 mol H2/mol glucose for C. saccharolyticus, T. neapolitana, and their co-culture respectively. Statistical analysis comparing the H2 production rate of the co-culture to either C. saccahrolyticus or T. neapolitana pure cultures indicated a significant difference in the H2 production rate (p < 0.05: t-test), with the highest rate of H2 production (36.02 mL L−1 h−1) observed from the co-culture fermentations. In order to monitor the presence of T. neapolitana in the bioprocess, we developed a qPCR method using 16S rRNA gene and hydrogenase (hydA) gene targets. The qPCR data using hydA primers specific to T. neapolitana showed an increase in hydA gene copies from 3.32 × 107 to 4.4 × 108hydA gene copies per mL confirming the influence of T. neapolitana in the synthetic consortium. acceptedVersion

Microbial electrosynthesis (MES) is a promising technology for the reduction of carbon dioxide into value-added multicarbon molecules. In order to broaden the product profile of MES processes, we developed a two-stage process for microbial conversion of carbon dioxide and electricity into long chain alkyl esters. In the first stage, the carbon dioxide is reduced to organic compounds, mainly acetate, in a MES process by Sporomusa ovata. In the second stage, the liquid end-products of the MES process are converted to the final product by a second microorganism, Acinetobacter baylyi in an aerobic bioprocess. In this proof-of-principle study, we demonstrate for the first time the bacterial production of long alkyl esters (wax esters) from carbon dioxide and electricity as the sole sources of carbon and energy. The process holds potential for the efficient production of carbon-neutral chemicals or biofuels. acceptedVersion

Some bacterial strains such as Komagataeibacter xylinus are able to produce cellulose as an extracellular matrix. In comparison to wood-based cellulose, bacterial cellulose (BC) holds interesting properties such as biodegradability, high purity, water-holding capacity, and superior mechanical and structural properties. Aiming toward improvement in BC production titer and tailored alterations to the BC film, we engineered K. xylinus to overexpress partial and complete bacterial cellulose synthase operon that encodes activities for BC production. The changes in cell growth, end metabolite, and BC production titers from the engineered strains were compared with the wild-type K. xylinus. Although there were no significant differences between the growth of wild-type and engineered strains, the engineered K. xylinus strains demonstrated faster BC production, generating 2–4-fold higher production titer (the highest observed titer was obtained with K. xylinus-bcsABCD strain producing 4.3 ± 0.46 g/L BC in 4 days). The mechanical and structural characteristics of cellulose produced from the wild-type and engineered K. xylinus strains were analyzed with a stylus profilometer, in-house built tensile strength measurement system, a scanning electron microscope, and an X-ray diffractometer. Results from the profilometer indicated that the engineered K. xylinus strains produced thicker BC films (wild type, 5.1 μm, and engineered K. xylinus strains, 6.2–10.2 μm). Scanning electron microscope revealed no principal differences in the structure of the different type BC films. The crystallinity index of all films was high (from 88.6 to 97.5%). All BC films showed significant piezoelectric response (5.0–20 pC/N), indicating BC as a promising sensor material. acceptedVersion

Background: Caloramator celer is a strict anaerobic, alkalitolerant, thermophilic bacterium capable of converting glucose to hydrogen (H2), carbon dioxide, acetate, ethanol and formate by a mixed acid fermentation. Depending on the growth conditions C. celer can produce H2 at high yields. For a biotechnological exploitation of this bacterium for H2 production it is crucial to understand the factors that regulate carbon and electron fluxes and therefore the final distribution of metabolites to channel the metabolic flux towards the desired product. Results: Combining experimental results from batch fermentations with genome analysis, reconstruction of central carbon metabolism and metabolic flux analysis (MFA), this study shed light on glucose catabolism of the thermophilic alkalitolerant bacterium C. celer. Two innate factors pertaining to culture conditions have been identified to significantly affect the metabolic flux distribution: culture pH and partial pressures of H2 (PH2). Overall, at alkaline to neutral pH the rate of biomass synthesis was maximized, whereas at acidic pH the lower growth rate and the less efficient biomass formation are accompanied with more efficient energy recovery from the substrate indicating high cell maintenance possibly to sustain intracellular pH homeostasis. Higher H2 yields were associated with fermentation at acidic pH as a consequence of the lower synthesis of other reduced by-products such as formate and ethanol. In contrast, PH2 did not affect the growth of C. celer on glucose. At high PH2 the cellular redox state was balanced by rerouting the flow of carbon and electrons to ethanol and formate production allowing unaltered glycolytic flux and growth rate, but resulting in a decreased H2 synthesis. Conclusion: C. celer possesses a flexible fermentative metabolism that allows redistribution of fluxes at key metabolic nodes to simultaneously control redox state and efficiently harvest energy from substrate even under unfavorable conditions (i.e. low pH and high PH2). With the H2 production in mind, acidic pH and low PH2 should be preferred for a high yield-oriented process, while a high productivity-oriented process can be achieved at alkaline pH and high PH2.

Halanaerobium saccharolyticum is a halophilic anaerobic fermentative bacterium capable of producing hydrogen, a potential future energy carrier molecule. The high-quality draft genome of H. saccharolyticum subsp. saccharolyticum strain DSM 6643 T consists of 24 contigs for 2,873,865 bp with a G+C content of 32.3%.

Background Wax esters are industrially relevant molecules exploited in several applications of oleochemistry and food industry. At the moment, the production processes mostly rely on chemical synthesis from rather expensive starting materials, and therefore solutions are sought from biotechnology. Bacterial wax esters are attractive alternatives, and especially the wax ester metabolism of Acinetobacter sp. has been extensively studied. However, the lack of suitable tools for rapid and simple monitoring of wax ester metabolism in vivo has partly restricted the screening and analyses of potential hosts and optimal conditions. Results Based on sensitive and specific detection of intracellular long-chain aldehydes, specific intermediates of wax ester synthesis, bacterial luciferase (LuxAB) was exploited in studying the wax ester metabolism in Acinetobacter baylyi ADP1. Luminescence was detected in the cultivation of the strain producing wax esters, and the changes in signal levels could be linked to corresponding cell growth and wax ester synthesis phases. Conclusions The monitoring system showed correlation between wax ester synthesis pattern and luminescent signal. The system shows potential for real-time screening purposes and studies on bacterial wax esters, revealing new aspects to dynamics and role of wax ester metabolism in bacteria.

Background Triacylglycerols are used in various purposes including food applications, cosmetics, oleochemicals and biofuels. Currently the main sources for triacylglycerol are vegetable oils, and microbial triacylglycerol has been suggested as an alternative for these. Due to the low production rates and yields of microbial processes, the role of metabolic engineering has become more significant. As a robust model organism for genetic and metabolic studies, and for the natural capability to produce triacylglycerol, Acinetobacter baylyi ADP1 serves as an excellent organism for modelling the effects of metabolic engineering for energy molecule biosynthesis. Results Beneficial gene deletions regarding triacylglycerol production were screened by computational means exploiting the metabolic model of ADP1. Four deletions, acr1, poxB, dgkA, and a triacylglycerol lipase were chosen to be studied experimentally both separately and concurrently by constructing a knock-out strain (MT) with three of the deletions. Improvements in triacylglycerol production were observed: the strain MT produced 5.6 fold more triacylglycerol (mg/g cell dry weight) compared to the wild type strain, and the proportion of triacylglycerol in total lipids was increased by 8-fold. Conclusions In silico predictions of beneficial gene deletions were verified experimentally. The chosen single and multiple gene deletions affected beneficially the natural triacylglycerol metabolism of A. baylyi ADP1. This study demonstrates the importance of single gene deletions in triacylglycerol metabolism, and proposes Acinetobacter sp. ADP1 as a model system for bioenergetic studies regarding metabolic engineering.

Background Pretreatment of lignocellulosic biomass can produce inhibitory compounds that are harmful for microorganisms used in the production of biofuels and other chemicals from lignocellulosic sugars. Selective inhibitor removal can be achieved with biodetoxification where microorganisms catabolize the inhibitors without consuming the sugars. We engineered the strictly aerobic Acinetobacter baylyi ADP1 for detoxification of lignocellulosic hydrolysates by removing the gene for glucose dehydrogenase, gcd, which catalyzes the first step in its glucose catabolism. Results The engineered A. baylyi ADP1 strain was shown to be incapable of consuming the main sugar components of lignocellulosic hydrolysates, i.e., glucose, xylose, and arabinose, but rapidly utilized acetate and formate. Formate was consumed during growth on acetate and by stationary phase cells, and this was enhanced in the presence of a common aromatic inhibitor of lignocellulosic hydrolysates, 4-hydroxybenzoate. The engineered strain tolerated glucose well up to 70 g/l, and the consumption of glucose, xylose, or arabinose was not observed in prolonged cultivations. The engineered strain was applied in removal of oxygen, a gaseous inhibitor of anaerobic fermentations. Co-cultivation with the A. baylyi ADP1 gcd knockout strain under initially aerobic conditions allowed the strictly anaerobic Clostridium butyricum to grow and produce hydrogen (H2) from sugars of the enzymatic rice straw hydrolysate. Conclusions We demonstrated that the model organism of bacterial genetics and metabolism, A. baylyi ADP1, could be engineered to be an efficient biodetoxification strain of lignocellulosic hydrolysates. Only one gene knockout was required to completely eliminate sugar consumption and the strain could be used in production of anaerobic conditions for the strictly anaerobic hydrogen producer, C. butyricum. Because of these encouraging results, we believe that A. baylyi ADP1 is a promising candidate for the detoxification of lignocellulosic hydrolysates for bioprocesses.