Ville Santala

Hydrogen production from glycerol using halophilic fermentative bacteria

Glycerol-based hydrogen production by the halophilic bacteria Halanaerobium saccharolyticum subspecies saccharolyticum and senegalensis was studied as batch experiments. The main metabolites of glycerol fermentation of both strains were hydrogen, carbon dioxide, and acetate. Subspecies saccharolyticum also produced 1,3-propanediol (1,3-PD), butyrate, and ethanol. The highest hydrogen yields were achieved with 2.5g/l glycerol and 150g/l salt at pH 7.4 (subsp. saccharolyticum, yield 0.6mol/mol glycerol) and at pH 7.0 (subsp. senegalensis, yield 1.6mol/mol glycerol). The hydrogen yield of subsp. senegalensis has potential for practical applications after scale-up and bioprocess optimizations and metabolic engineering after genome-wide sequencing could be applied to improve the yield of subsp. saccharolyticum.

Improved Triacylglycerol Production in Acinetobacter baylyi ADP1 by Metabolic Engineering

BackgroundTriacylglycerols 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.ResultsBeneficial 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.ConclusionsIn 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.

1,3-Propanediol production and tolerance of a halophilic fermentative bacterium, Halanaerobium saccharolyticum subsp. saccharolyticum.

1,3-Propanediol (1,3-PD) is widely used in polymer industry in production of polyethers, polyesters and polyurethanes. In this article, a study on 1,3-PD production and tolerance of Halanaerobium saccharolyticum subsp. saccharolyticum is presented. 1,3-PD production was optimized for temperature, vitamin B(12) and acetate concentration. The highest 1,3-PD concentrations and yields (0.6 mol/mol glycerol) were obtained at vitamin B₁₂ concentration 64 μg/l and an inverse correlation between 1,3-PD and hydrogen production was observed with varying vitamin B₁₂ concentrations. In the studied temperature range and initial acetate concentrations up to 10 g/l, no significant variations were observed in 1,3-PD production. High initial acetate (29-58 g/l) was observed to cause slight decrease in 1,3-PD concentrations produced but no effects on 1,3-PD yields (mol/mol glycerol). Initial 1,3-PD concentrations inhibited the growth of H. saccharolyticum subsp. saccharolyticum. When initial 1,3-PD concentration was raised from 1g/l to 57 g/l, a decrease of 12% to 75%, respectively, in the highest optical density was observed.

Biohydrogen production in alkalithermophilic conditions: Thermobrachium celere as a case study.

In the present work the hydrogenesis in the anaerobic alkalithermophilic bacterium Thermobrachium celere was studied. The impact of several factors on hydrogen production during glucose fermentation was investigated in batch conditions. The optimal hydrogen production occurred at pH (67 °C) 8.2 with phosphate buffer concentration of 50 mM. Hydrogen yield reached the highest value of 3.36 mol H2/mol glucose when the partial pressure in the gas headspace was reduced. Supplementation of nitrogen sources and iron affected hydrogen production. Under optimized conditions, the maximum H2 accumulation and H2 production rate were estimated to be respectively 124.3 mmol H2/l culture and 20.7 mmol H2/l/h. Considering the efficient and rapid hydrogen evolution, and the ability to grow in extreme environments, T. celere might be a good candidate for biohydrogen production in open (non-sterile) bioprocess system.

Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation

In microbial ecosystems, bacteria are dependent on dynamic interspecific interactions related to carbon and energy flow. Substrates and end-metabolites are rapidly converted to other compounds, which protects the community from high concentrations of inhibitory molecules. In biotechnological applications, pure cultures are preferred because of the more straight-forward metabolic engineering and bioprocess control. However, the accumulation of unwanted side products can limit the cell growth and process efficiency. In this study, a rationally engineered coculture with a carbon channeling system was constructed using two well-characterized model strains Escherichia coli K12 and Acinetobacter baylyi ADP1. The directed carbon flow resulted in efficient acetate removal, and the coculture showed symbiotic nature in terms of substrate utilization and growth. Recombinant protein production was used as a proof-of-principle example to demonstrate the coculture utility and the effects on product formation. As a result, the biomass and recombinant protein titers of E. coli were enhanced in both minimal and rich medium simple batch cocultures. Finally, harnessing both the strains to the production resulted in enhanced recombinant protein titers. The study demonstrates the potential of rationally engineered cocultures for synthetic biology applications.

Real-Time monitoring of intracellular wax ester metabolism

BackgroundWax 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.ResultsBased 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.ConclusionsThe 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.

Engineering and Characterization of Bacterial Nanocellulose Films as Low Cost and Flexible Sensor Material

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.

Bidirectional fluorescence resonance energy transfer (FRET) in mutated and chemically modified yellow fluorescent protein (YFP).

Fluorescence resonance energy transfer (FRET) using fluorescent protein variants are used for studying the associations and biomolecular motions of macromolecules inside the cell. Intramolecular FRET utilizing fluorescent chemical labels has been applied in nucleic acid chemistry for detection of specific sequence. However, the biotechnological applications of intramolecular FRET in fluorescent proteins have not been exploited. This study demonstrates the intramolecular FRET between fluorescent protein and conjugated chemical label whereby FRET occurs from inside to outside and vice versa for fluorescent protein. The fluorescent protein is modified for the attachment of chemical fluorophores and the novel FRET pairs created by conjugation are MDCC (435/475)-Citrine (516/529) and Citrine-Alexa fluor (568/603). These protein-label pairs exhibited strong intramolecular FRET and the energy transfer efficiency was determined based on the time evolution of the ratio of emission intensities of labeled and unlabeled proteins. The efficiency was found to be 0.79 and 0.89 for MDCC-Citrine and 0.24 and 0.65 for Citrine-Alexa Fluor pairs when the label is conjugated at different sites in the protein. Förster distance and the average distance between the fluorophores were also determined. The bidirectional approach described here can provide new insights into designing FRET-based sensors.

Production of long chain alkyl esters from carbon dioxide and electricity by a two-stage bacterial process

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.

Metabolic Engineering of Acinetobacter baylyi ADP1 for Improved Growth on Gluconate and Glucose

ABSTRACT A high growth rate in bacterial cultures is usually achieved by optimizing growth conditions, but metabolism of the bacterium limits the maximal growth rate attainable on the carbon source used. This limitation can be circumvented by engineering the metabolism of the bacterium. Acinetobacter baylyi has become a model organism for studies of bacterial metabolism and metabolic engineering due to its wide substrate spectrum and easy-to-engineer genome. It produces naturally storage lipids, such as wax esters, and has a unique gluconate catabolism as it lacks a gene for pyruvate kinase. We engineered the central metabolism of A. baylyi ADP1 more favorable for gluconate catabolism by expressing the pyruvate kinase gene (pykF) of Escherichia coli. This modification increased growth rate when cultivated on gluconate or glucose as a sole carbon source in a batch cultivation. The engineered cells reached stationary phase on these carbon sources approximately twice as fast as control cells carrying an empty plasmid and produced similar amount of biomass. Furthermore, when grown on either gluconate or glucose, pykF expression did not lead to significant accumulation of overflow metabolites and consumption of the substrate remained unaltered. Increased growth rate on glucose was not accompanied with decreased wax ester production, and the pykF-expressing cells accumulated significantly more of these storage lipids with respect to cultivation time.