Hydrogen production in an aerobic environment by Escherichia coli K-12

Escherichia coli (E. coli) is a facultative anaerobe that can grow in a variety of environmental conditions. In the complete absence of O2, E. coli can perform a mixed-acid fermentation that contains within it an elaborate metabolism of formic acid. In this study, we use cavity-enhanced Raman spectroscopy (CERS), FTIR, liquid Raman spectroscopy, isotopic labelling, and molecular genetics to make advances in the understanding of bacterial formate and H2 metabolism. It is shown that, under anaerobic conditions, formic acid is generated endogenously, excreted briefly from the cell, and then taken up again to be disproportionated to H2 and CO2 by formate hydrogenlyase (FHL-1). However, exogenously added D-labelled formate behaves quite differently from the endogenous formate and is taken up immediately, independently, and possibly by a different mechanism, by the cell and converted to H2 and CO2. Our data support an anion-proton symport model for formic acid transport. In addition, when E. coli was grown in a microaerobic environment it was possible to analyse aspects of formate and O2 respiration occurring alongside anaerobic metabolism. While cells growing under microaerobic conditions generated endogenous formic acid, no H2 was produced. However, addition of exogenous formate at the outset of cell growth did induce FHL-1 biosynthesis and resulted in formate-dependent H2 production in the presence of O2.

Antibiofilm Effects of Heated Scallop Shell Powder on Campylobacter jejuni Biofilms

Methods to reuse large numbers of scallop shells from the harvesting regions of Japan are being explored. The major component of scallop shells is calcium carbonate (CaCO3), which forms the powerful bactericidal agent, calcium oxide (CaO), when heated. Heated scallop shell powder (HSSP) exhibits strong and broad-spectrum antimicrobial activity against bacteria, fungi, and viruses. This study investigated the antibiofilm activity of HSSP against the biofilms of Campylobacter jejuni, which is the predominant species in campylobacteriosis. Biofilm samples of C. jejuni were prepared on 0.45 µm filter paper under microaerobic conditions. The HSSP treatment inactivated and eradicated C. jejuni biofilms. The resistance of C. jejuni biofilms to HSSP was significantly higher than that of the floating cells. Moreover, the antibiofilm activity of the HSSP treatment against C. jejuni biofilms was higher than that of NaOH treatment at the same pH. These results indicated that HSSP treatment is an effective method for controlling C. jejuni biofilms.

Enhanced production of Shiga toxin 1 in enterohaemorrhagic Escherichia coli by oxygen

Enterohaemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Although stx1 and stx2 were found within the late operons of the Stx-encoding phages (Stx-phages), stx1 could mainly be transcribed from the stx1 promoter (P Stx1), which represents the functional operator-binding site (Fur box) for the transcriptional regulator Fur (ferric uptake regulator), upstream of stx1. In this study, we found that the production of Stx1 by EHEC was affected by oxygen concentration. Increased Stx1 production in the presence of oxygen is dependent on Fur, which is an Fe2+-responsive transcription factor. The intracellular Fe2+ pool was lower under microaerobic conditions than under anaerobic conditions, suggesting that lower Fe2+ availability drove the formation of less Fe2+-Fur, less DNA binding to the P Stx1 region, and an increase in Stx1 production.

Co-Production of Isoprene and Lactate by Engineered Escherichia coli in Microaerobic Conditions

Lactate and isoprene are two common monomers for the industrial production of polyesters and synthetic rubbers. The present study tested the co-production of D-lactate and isoprene by engineered Escherichia coli in microaerobic conditions. The deletion of alcohol dehydrogenase (adhE) and acetate kinase (ackA) genes, along with the supplementation with betaine, improved the co-production of lactate and isoprene from the substrates of glucose and mevalonate. In fed-batch studies, microaerobic fermentation significantly improved the isoprene concentration in fermentation outlet gas (average 0.021 g/L), compared with fermentation under aerobic conditions (average 0.0009 g/L). The final production of D-lactate and isoprene can reach 44.0 g/L and 3.2 g/L, respectively, through fed-batch microaerobic fermentation. Our study demonstrated a dual-phase production strategy in the co-production of isoprene (gas phase) and lactate (liquid phase). The increased concentration of gas-phase isoprene could benefit the downstream process and decrease the production cost to collect and purify the bio-isoprene from the fermentation outlet gas. The proposed microaerobic process can potentially be applied in the production of other volatile bioproducts to benefit the downstream purification process.

Waterborne Isolates of Campylobacter jejuni Are Able to Develop Aerotolerance, Survive Exposure to Low Temperature, and Interact With Acanthamoeba polyphaga

Campylobacter jejuni is regarded as the leading cause of bacterial gastroenteritis around the world. Even though it is generally considered to be a sensitive microaerobic pathogen, it is able to survive in the environment outside of the intestinal tract of the host. This study aimed to assess the impact of selected environmental parameters on the survival of 14 C. jejuni isolates of different origins, including 12 water isolates. The isolates were tested for their antibiotic resistance, their ability to survive at low temperature (7°C), develop aerotolerance, and to interact with the potential protozoan host Acanthamoeba polyphaga. The antibiotic susceptibility was determined by standard disk diffusion according to EUCAST. Out of the 14 isolates, 8 were resistant to ciprofloxacin (CIP) and 5 to tetracycline (TET), while only one isolate was resistant to erythromycin (ERY). Five isolates were resistant to two different antibiotic classes. Tetracycline resistance was only observed in isolates isolated from wastewater and a clinical sample. Further, the isolates were tested for their survival at 7°C under both aerobic and microaerobic conditions using standard culture methods. The results showed that under microaerobic conditions, all isolates maintained their cultivability for 4 weeks without a significant decrease in the numbers of bacteria and variation between the isolates. However, significant differences were observed under aerobic conditions (AC). The incubation led to a decrease in the number of cultivable cells, with complete loss of cultivability after 2 weeks (one water isolate), 3 weeks (7 isolates), or 4 weeks of incubation (6 isolates). Further, all isolates were studied for their ability to develop aerotolerance by repetitive subcultivation under microaerobic and subsequently AC. Surprisingly, all isolates were able to adapt and grow under AC. As the last step, 5 isolates were selected to evaluate a potential protective effect provided by A. polyphaga. The cocultivation of isolates with the amoeba resulted in the survival of about 40% of cells treated with an otherwise lethal dose of gentamicin. In summary, C. jejuni is able to adapt and survive in a potentially detrimental environment for a prolonged period of time, which emphasizes the role of the environmental transmission route in the spread of campylobacteriosis.

Oligo-heterotrophic activity of Marinobacter subterrani creates an indirect Fe(II)-oxidation phenotype in gradient tubes

Autotrophic bacteria utilizing Fe(II) as their energy and electron sources for growth affect multiple biogeochemical cycles. Some chemoheterotrophic bacteria have also been considered to exhibit an Fe(II) oxidation phenotype. For example, several Marinobacter strains have been reported to oxidize Fe(II) based on formation of oxidized iron bands in semi-solid gradient tubes that produce opposing concentration gradients of Fe(II) and oxygen. While gradient tubes are a simple and visually compelling method to test for Fe(II) oxidation, this method alone cannot confirm if, and to what extent, Fe(II) oxidation is linked to metabolism in chemoheterotrophic bacteria. Here we probe the possibility of protein-mediated and metabolic byproduct-mediated Fe(II) oxidation in Marinobacter subterrani JG233, a chemoheterotroph previously proposed to oxidize Fe(II). Results from conditional and mutant studies, along with measurements of Fe(II) oxidation rates suggest M. subterrani is unlikely to facilitate Fe(II) oxidation under microaerobic conditions. We conclude that the Fe(II) oxidation phenotype observed in gradient tubes inoculated with M. subterrani JG233 is a result of oligo-heterotrophic activity, shifting the location where oxygen dependent chemical Fe(II) oxidation occurs, rather than a biologically-mediated process. Importance Gradient tubes are the most commonly used method to isolate and identify neutrophilic Fe(II)-oxidizing bacteria. The formation of oxidized iron bands in gradient tubes provides a compelling assay to ascribe the ability to oxidize Fe(II) to autotrophic bacteria whose growth is dependent on Fe(II) oxidation. However, the physiological significance of Fe(II) oxidation in chemoheterotrophic bacteria is less well understood. Our work suggests that oligo-heterotrophic activity of certain bacteria may create a false-positive phenotype in gradient tubes by altering the location of the abiotic, oxygen-mediated oxidized iron band. Based on the results and analysis presented here, we caution against utilizing gradient tubes as the sole evidence for the capability of a strain to oxidize Fe(II) and that additional experiments are necessary to ascribe this phenotype to new isolates.

“Take It or Leave It”—Factors Regulating Competence Development and DNA Uptake in Campylobacter jejuni

Campylobacter jejuni has a large adaptive potential due to enormous genetic exchange. Factors regulating natural transformation in this food-borne pathogen are largely unknown but of interest for the application of sustained reduction strategies in the food-processing industry. Using a single cell DNA uptake assay, we visualized that recognition of methylated C. jejuni DNA was essential for the first step of DNA uptake into a DNase resistant state. Transformation rates using a resistance marker correlated with the fraction of competent bacteria, harboring one to maximally four locations of active DNA uptake, not necessarily being located at the cell pole. Competence developed with rising pH between 6.5 and 7.5 under microaerobic conditions and was nearly insensitive towards growth temperatures between 32 °C and 42 °C, CO2 concentrations ranging from 0 to 50% and growth rates. However, competence development was abolished at pH 5 or under aerobic stress conditions, in which the bacteria ceased growth but fully survived. The DNA uptake machinery in competent bacteria shut down at slightly acidic pH and was reversibly switched on upon neutralization. It was dependent on the proton motive force and, in contrast to competence development, slightly enhanced under aerobic conditions. The results suggest that natural transformation in C. jejuni occurs in the neutral and microaerobic intestinal environment for enhanced genetic diversity and pre-adaption before host switch. In addition, highly competent bacteria might be shed into the environment, still able to acquire genetic material for increased survival.

Microaerobic Digestion of Low-Biodegradable Sewage Sludge: Effect of Air Dosing in Batch Reactors

The adoption of prolonged solid retention times during the biological treatment of urban wastewaters is a well-known strategy to reduce sewage sludge production. However, it also results in the production of a biological sludge with low percentages of biodegradable organic matter, also characterized by high humification degrees, which may hamper the anaerobic digestion treatment aimed at sludge stabilization. To accelerate the hydrolytic stage, the application of microaerobic conditions during the anaerobic digestion of low-biodegradable sewage sludge was investigated in this study. In particular, six bio-methanation tests of a real sewage sludge were carried out, introducing air in the bioreactors with doses ranging between 0 and 16.83 L air/kg VSin d, in order to evaluate the air dosage that optimizes the biomethane production and organic matter degradation. Notably, the lower air loading rates investigated in this study, such as 0.68 and 1.37 L air/kg VSin d, led to an increase in methane production of up to 19%, due to a higher degradation of total lipids and proteins. In addition, these microaerobic conditions also resulted in a decrease in the sludge humification degree and in lower volatile fatty acid accumulation.

Comparison of Spathaspora passalidarum and recombinant Saccharomyces cerevisiae for integration of first- and second-generation ethanol production

ABSTRACT First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G–E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2 and XKS1 genes and a mutant ([4–59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G–E2G production process.