Potential for Increased Human Foodborne Exposure to PCDD/F When Recycling Sewage Sludge on Agricultural Land

Sewage sludge from municipal wastewater treatment is used in agriculture as a nutrient source and to aid in moisture retention. To examine the potential impact of sludge-amended soil on exposures to polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) from plant and animal foods, we conducted a review of published empirical data from international sources. Levels of PCDD/F in municipal sewage sludge ranged from 0.0005 to 8,300 pg toxic equivalents (TEQ)/g. Background levels in soil ranged from 0.003 to 186 pg TEQ/g. In sludge-amended soils, levels of PCDD/F ranged from 1.4 to 15 pg TEQ/g. Studies that measured levels before and after sludge treatment showed an increase in soil concentration after treatment. Relationships between PCDD/F levels in soil and resulting concentrations in plants were very weakly positive for unpeeled root crops, leafy vegetables, tree fruits, hay, and herbs. Somewhat stronger relationships were observed for plants of the cucumber family. In all cases, large increases in soil concentration were required to achieve a measurable increase in plant contamination. A considerably stronger positive relationship was observed between PCDD/F in feed and resulting levels in cattle tissue, suggesting bioaccumulation. Although PCDD/Fs are excreted in milk, no association was found between feed contamination and levels of PCDD/Fs measured in milk. There is a paucity of realistic data describing the potential for entry of PCDD/Fs into the food supply via sewage sludge. Currently available data suggest that sewage sludge application to land used for most crops would not increase human exposure. However, the use of sludge on land used to graze animals appears likely to result in increased human exposure to PCDD/F

Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers

An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige–Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T0 generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T1 seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T1 generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use

Batch and continuous culture-based selection strategies for acetic acid tolerance in xylose-fermenting Saccharomyces cerevisiae

Acetic acid tolerance of Saccharomyces cerevisiae is crucial for the production of bioethanol and other bulk chemicals from lignocellulosic plant-biomass hydrolysates, especially at a low pH. This study explores two evolutionary engineering strategies for the improvement of acetic acid tolerance of the xylose-fermenting S. cerevisiae RWB218, whose anaerobic growth on xylose at pH 4 is inhibited at acetic acid concentrations >1 g L(-1) : (1) sequential anaerobic, batch cultivation (pH 4) at increasing acetic acid concentrations and (2) prolonged anaerobic continuous cultivation without pH control, in which acidification by ammonium assimilation generates selective pressure for acetic acid tolerance. After c. 400 generations, the sequential-batch and continuous selection cultures grew on xylose at pH≤4 with 6 and 5 g L(-1) acetic acid, respectively. In the continuous cultures, the specific xylose-consumption rate had increased by 75% to 1.7 g xylose g(-1) biomass h(-1) . After storage of samples from both selection experiments at -80 °C and cultivation without acetic acid, they failed to grow on xylose at pH 4 in the presence of 5 g L(-1) acetic acid. Characterization in chemostat cultures with linear acetic acid gradients demonstrated an acetate-inducible acetic acid tolerance in samples from the continuous selection protocol

Transfer pathways of PCDD/PCDF to fruits

In a field experiment the transfer of PCDD/PCDF from contaminated soils to apples and pears was investigated. The soil PCDD/PCDF contamination differed in total PCDD/PCDF content and in the homologue pattern. PCDD/PCDF content in whole fruits ranged from 1-4 ng/kg FW. Soil PCDD/PCDF contamination was not correlated with the PCDD/PCDF concentrations of the fruits. The higher PCDD/PCDF concentrations of the peel in comparison to the pulp indicate that air-borne PCDD/PCDF is the major pathway for fruit contamination. Therefore peeling of fruits can possibly reduce PCDD PCDF intake

Transfer of PCDD/PCDF from contaminated soils into carrots, lettuce and peas

In a field experiment, the PCDD/PCDF transfer pathways from soil into carrots, lettuce and peas has been investigated. PCDD/PCDF contamination levels in soil varied between 5 ng I-TEq/kg on the control plot and 56 ng I-TEq/kg on the contaminated plot. PCDD/PCDF levels in carrots were threefold higher in the contaminated plot than in the control plot, which was a result of a tenfold increase in the PCDD/PCDF levels of the peel. PCDD/PCDF levels in lettuce and peas were not higher when grown on the contaminated plot and were much lower than in carrots, which indicates that the PCDD/PCDF in lettuce and peas from both plots are of atmospheric origin

Key Process Conditions for Production of C4 Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae Strain▿

A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter−1 of malate at a yield of 0.42 mol (mol glucose)−1 in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO3, was required for efficient C4 dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO3 dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose)−1, whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered S. cerevisiae strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO2, calcium, and O2). Under optimized conditions, a malate yield of 0.48 ± 0.01 mol (mol glucose)−1 was obtained in bioreactors, a 19% increase over yields in shake flask experiments

Quantitative Physiology of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates

Growth at near-zero specific growth rates is a largely unexplored area of yeast physiology. To investigate the physiology of Saccharomyces cerevisiae under these conditions, the effluent removal pipe of anaerobic, glucose-limited chemostat culture (dilution rate, 0.025 h–1) was fitted with a 0.22-µm-pore-size polypropylene filter unit. This setup enabled prolonged cultivation with complete cell retention. After 22 days of cultivation, specific growth rates had decreased below 0.001 h–1 (doubling time of >700 h). Over this period, viability of the retentostat cultures decreased to ca. 80%. The viable biomass concentration in the retentostats could be accurately predicted by a maintenance coefficient of 0.50 mmol of glucose g–1 of biomass h–1 calculated from anaerobic, glucose-limited chemostat cultures grown at dilution rates of 0.025 to 0.20 h–1. This indicated that, in contrast to the situation in several prokaryotes, maintenance energy requirements in S. cerevisiae do not substantially change at near-zero specific growth rates. After 22 days of retentostat cultivation, glucose metabolism was predominantly geared toward alcoholic fermentation to meet maintenance energy requirements. The strict correlation between glycerol production and biomass formation observed at higher specific growth rates was not maintained at the near-zero growth rates reached in the retentostat cultures. In addition to glycerol, the organic acids acetate, D-lactate, and succinate were produced at low rates during prolonged retentostat cultivation. This study identifies robustness and by-product formation as key issues in attempts to uncouple growth and product formation in S. cerevisiae. Erratum: http://aem.asm.org/cgi/content/full/75/23/757

Long-Term Adaptation of Saccharomyces cerevisiae to the Burden of Recombinant Insulin Production

High-level production of heterologous proteins is likely to impose a metabolic burden on the host cell and can thus affect various aspects of cellular physiology. A data-driven approach was applied to study the secretory production of a human insulin analog precursor (IAP) in Saccharomyces cerevisiae during prolonged cultivation (80 generations) in glucose-limited aerobic chemostat cultures. Physiological characterization of the recombinant cells involved a comparison with cultures of a congenic reference strain that did not produce IAP, and time-course analysis of both strains aimed at identifying the metabolic adaptation of the cells towards the burden of IAP production. All cultures were examined at high cell density conditions (30g/L dry weight) to increase the industrial relevance of the results. The burden of heterologous protein production in the recombinant strain was explored by global transcriptome analysis and targeted metabolome analysis, including the analysis of intracellular amino acid pools, glycolytic metabolites, and TCA intermediates. The cellular re-arrangements towards IAP production were categorized in direct responses, for example, enhanced metabolism of amino acids as precursors for the formation of IAP, as well as indirect responses, for example, changes in the central carbon metabolism. As part of the long-term adaptation, a metabolic re-modeling of the IAP-expressing strain was observed, indicating an augmented negative selection pressure on glycolytic overcapacity, and the emergence of mitochondrial dysfunction. The evoked metabolic re-modeling of the cells led to less optimal conditions with respect to the expression and processing of the target protein and thus decreased the cellular expression capacity for the secretory production of IAP during prolonged cultivation