Rapid prototyping of microbial production strains for the biomanufacture of potential materials monomers

Bio-based production of industrial chemicals using synthetic biology can provide alternative green routes from renewable resources, allowing for cleaner production processes. To efficiently produce chemicals on-demand through microbial strain engineering, biomanufacturing foundries have developed automated pipelines that are largely compound agnostic in their time to delivery. Here we benchmark the capabilities of a biomanufacturing pipeline to enable rapid prototyping of microbial cell factories for the production of chemically diverse industrially relevant material building blocks. Over 85 days the pipeline was able to produce 17 potential material monomers and key intermediates by combining 160 genetic parts into 115 unique biosynthetic pathways. To explore the scale-up potential of our prototype production strains, we optimized the enantioselective production of mandelic acid and hydroxymandelic acid, achieving gram-scale production in fed-batch fermenters. The high success rate in the rapid design and prototyping of microbially-produced material building blocks reveals the potential role of biofoundries in leading the transition to sustainable materials production.     

Rapid assessment of the microbial deterioration of Polyurethanes

An account is presented of experimental procedures designed to evaluate rapidly the susceptibility of polyurethane formulations to microbial attack.The effect of stress upon the susceptibility of polyester polyurethane degradation is examined and evidence is presented which suggests that stressing increases degradation significantly.In vitro tests are outlined which give rapid qualitative assays of the susceptibility of polyurethane formulations to microbial attack.     

Rapid milk progesterone assay as a tool for the selection of potential donor cows prior to superovulation

This study investigated the accuracy of a commercially available rapid milk progesterone (P(4)) assay (RMPA) and its usefulness for the screening of potential donor cows prior to superovulatory treatment. Superovulation was induced in 90 lactating Holstein-Friesian crossbred dairy cows with twice daily injections over a 4-day period for a total of 40 mg follicle stimulating hormone (FSH-P), starting 9 to 13 d post estrus. Prior to induction of superovulation, a milk sample was collected and assayed for a P(4) level using the RMPA. The test determines P(4) by a simple visual color inspection of the respective sample, which is compared to a standard containing 10.5 ng/ml of P(4). All animals were divided into six groups according to the color intensity of their sample; three groups had a lower level, one group had an equal level and two groups had a higher P(4) level than the standard. Results of the semiquantitative RMPA were verified by a quantitative enzymeimmunoassay (EIA). Samples evaluated as equivalent to the standard had a mean P(4) level of 10.7 +/- 1.3 ng/ml (x +/- SEM). In total, P(4) levels differed (P<0.05) among groups, except in those with lower P(4) concentrations (1.1 +/- 0.0; 1.0 +/- 0.0; 3.7 +/- 1.5; 10.7 +/- 1.3; 13.8 +/- 1.3; 19.0 +/- 1.5 ng/ml, respectively). The correlation between RMPA-groups and EIA P(4) levels was 0.69 (P<0.001). Donors classified as having less P(4) than the standard yielded fewer corpora lutea (CL) (P<0.005), ova and embryos (P<0.05), and transferable embryos (P<0.05) compared with donors having similar or higher P(4) levels (3.4 +/- 1.0 vs 10.8 +/- 0.7 CL; 1.7 +/- 0.8 vs 6.2 +/- 0.9 ova and embryos; 1.2 +/- 0.7 vs 2.8 +/- 0.4 transferable embryos). Our results indicate that RMPA determines milk P(4) levels with sufficient accuracy and is a simple and useful tool for the screening of potential donor cows.     

A simple method to evaluate the potential for degradation of antifouling biocides

A simple method to measure the degradation of antifouling biocides is described which measures the loss of biocidal activity from seawater by bioassay. The bioassay employs either the ship‐fouling diatom Amphora or the brine shrimp Anemia. Loss of bioaclivity from sterile seawater indicates abiotic degradation whilst loss of bioactivity from natural seawater indicates biodegradation. Results are presented for three biocides, viz. the trihalomethylthio compound, N‐dichlorofluoromethylthio‐N’,N'‐dimethyl‐N‐phenyl‐sulphamide (Preventol A4S), di‐n‐octylamine, and the isothiazolone compound 4,5‐dichloro‐2‐n‐octyl‐4‐isothiazolin‐3‐one (Sea‐Nine 211).     

Degradation of polyhydroxyalkanoates and the composition of microbial destructors under natural conditions

The degradation dynamics of polyhydroxyalkanoates of different composition has been studied in an eutrophic storage reservoir for two seasons. It has been shown that the biodegradation of polymers under natural conditions depends not only on their structure and physicochemical properties but also, to a great extent, on a complex of weather-climatic conditions affecting the state of the reservoir ecosystem. The molecular genetic analysis of 16S rRNA has revealed bacterial species (clones) probably involved in the degradation of polyhydroxyalkanoates in a model storage reservoir.     

Degradation of polyhydroxyalkanoates and the composition of microbial destructors under natural conditions

The degradation dynamics of polyhydroxyalkanoates of different composition has been studied in an eutrophic storage reservoir for two seasons. It has been shown that the biodegradation of polymers under natural conditions depends not only on their structure and physicochemical properties but also, to a great extent, on a complex of weather-climatic conditions affecting the state of the reservoir ecosystem. The molecular genetic analysis of 16S rRNA has revealed bacterial species (clones) probably involved in the degradation of polyhydroxyalkanoates in a model storage reservoir.     

A novel assay for the distribution of pyrithione biocides in bacterial cells

Sodium pyrithione and zinc pyrithione (NaPT and ZnPT, respectively) are widely used as cosmetic preservatives and metal chelating agents. They are commonly assayed using thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). However, a simple quantitative colorimetric assay has not been previously reported for these compounds. This paper describes the development of a spectrophotometric assay for the quantification of the pyrithiones which is based on the chelation of copper (II) ions by the biocides. This assay was developed in order to facilitate the determination of the distribution of these biocides in the Gram-negative bacteria Escherichia coli NCIMB 10000 and Pseudomonas aeruginosa NCIMB 10548. Sodium pyrithione was exhibited only in the cytosol of E. coli and Ps. aeruginosa . Zinc pyrithione, however, was assayed in the cytosol of both bacteria and was found in the cell envelope of Ps. aeruginosa . These findings suggest that the pyrithione biocides are active within bacterial cells as well as at the cell membrane.     

Contamination level and potential health risk assessment of hexavalent chromium in soils from a coal chemical industrial area in Northwest China

Abstract

This study aimed to evaluate the total Cr and Cr(VI) contamination levels and assess the possible health risk of Cr(VI) in soils from a coal chemical industrial area in Northwest China. The contamination factor (CF) was used to calculate the total Cr and Cr(VI) contamination levels in soils from the study area. The highest concentration of Cr(VI) (69.58?mg/kg) was found in the top soil (0–20?cm) with the distance of 10 m to the coal cinder heap. The carcinogenic risk (CR) and hazard quotient (HQ) were estimated for health risk to workers by using “Chinese Technical Guidelines for Risk Assessment of Contaminated Sites (HJ 25.3-2014).” The results showed that the soils from the study area were moderately polluted by the total Cr and Cr(VI). There was no serious non-CR (HQ < 1). However, the CR values of Cr(VI) were significantly higher than the threshold value, indicating that workers are facing serious threat of Cr(VI). Inhalation (70.32%) was the main exposure pathway to CR, followed by dermal contact (20.64%), and then ingestion (9.04%). These results provide basic information of Cr(VI) pollution control and environment management in coal chemical industrial areas.     

A qauntitative in vitro assay for the evaluation of phototoxic potential of topically applied materials

A quantitative in vitro method for phototoxic evaluation of chemicals has been developed and validated. The assay uses Saccharomyces cerevisiae, seeded in an agar overlay on top of a plate count agar base. 8-Methoxy psoralen is used as a reference standard against which materials are measured. Activity is quantified by cytotoxicity measured as zones of inhibition. Several known phototoxins (heliotropine, lyral, phantolid, and bergamot oil) and photoallergens (6-methyl coumarin and musk ambrette) are used to validate the assay. An excellent correlation is observed between in vivo studies employing Hartley albino guinea pigs and the in vitro assay for several fragrance raw materials and other chemicals. The in vitro assay exhibits a greater sensitivity from 2–500 fold. For three fragrance oils, the in vitro assay detects low levels of photobiological activity while the in vivo assay is negative. Although the in vitro assay does not discriminate between phototoxins and photoallergens, it can be used for screening of raw materials so that reduction in animal usage can be achieved while maintaining the protection of the consumer.Abbreviations C centigrade - cm centimeter - cm² square centimeter - ml milliliters - mm millimeters - 8-MOP 8-methoxy psoralen - mW milliwatt - nm nanometer - UVA ultraviolet radiation, 320–400 nm The first in a series of research papers on alternatives to animal toxicity studies.     

Optimisation of a potency assay for the assessment of immunomodulative potential of clinical grade multipotent mesenchymal stromal cells

Clinical use of multipotent Mesenchymal Stromal Cell (MSC)-based medicinal products requires their production in compliance with Good Manufacturing Practices, thus ensuring that the final drug product meets specifications consistently from batch to batch in terms of cell viability, identity, purity and potency. Potency relates to the efficacy of the medicine in its target clinical indication, so adequate release tests need to be defined and validated as quality controls. Herein we report the design and optimisation of parameters affecting the performance of an in vitro cell-based assay for assessing immunomodulatory potential of clinical grade MSC for human use, based on their capacity to inhibit proliferation of T lymphocytes under strong polyclonal stimuli. The resulting method was demonstrated to be reproducible and relatively simple to execute. Two case studies using clinical grade MSC are presented as examples to illustrate the applicability of the methodology described in this work.     

Isolation of lactic acid bacteria from Malaysian foods and assessment of the isolates for industrial potential

Two traditional fermented food 'tapai' (fermented tapioca) and 'tempoyak' (fermented durian flesh), chilli puree and fresh goat's milk were used as sources for the isolation of lactic acid bacteria (LAB). A total of 126 isolates were obtained and by sequential screening for catalase activity and Gram-staining, 55 were determined to be LAB out of which 16 were established to be homofermentative by the gel plug test. Seven isolates were identified by use of the API 50CHL kit and two lactobacilli strains and one lactococci strain were selected to study their growth and lactic acid production profiles in a time course experiment. The lactobacilli strains, both isolated from 'tapai', produced higher amounts of cells and lactic acid from glucose as compared to the lactococci strain isolated from fresh goat's milk.     

Bacterial assay for the rapid assessment of antifouling and fouling release properties of coatings and materials

An assay has been developed to accurately quantify the growth and release behaviour of bacterial biofilms on several test reference materials and coatings, using the marine bacterium Cobetia marina as a model organism. The assay can be used to investigate the inhibition of bacterial growth and release properties of many surfaces when compared to a reference. The method is based upon the staining of attached bacterial cells with the nucleic acid-binding, green fluorescent SYTO 13 stain. A strong linear correlation exists between the fluorescence of the bacterial suspension measured (RFU) using a plate reader and the total bacterial count measured with epifluorescence microscopy. This relationship allows the fluorescent technique to be used for the quantification of bacterial cells attached to surfaces. As the bacteria proliferate on the surface over a period of time, the relative fluorescence unit (RFU) measured using the plate reader also shows an increase with time. This was observed on all three test surfaces (glass, Epikote and Silastic T2) over a period of 4 h of bacterial growth, followed by a release assay, which was carried out by the application of hydrodynamic shear forces using a custom-made rotary device. Different fixed rotor speeds were tested, and based on the release analysis, 12 knots was used to provide standard shear force. The assay developed was then applied for assessing three different antifouling coatings of different surface roughness. The novel assay allows the rapid and sensitive enumeration of attached bacteria directly on the coated surface. This is the first plate reader assay technique that allows estimation of irreversibly attached bacterial cells directly on the coated surface without their removal from the surface or extraction of a stain into solution.     

Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay

In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min.     

Fermentation characteristics of exopolysaccharide-producing lactic acid bacteria from sourdough and assessment of the isolates for industrial potential

Lactic acid bacteria (LAB) with antimicrobial activity and high exopolysaccharide (EPS) production ability isolated from sourdough were studied for their fermentation characteristics as potential new starter cultures. The values of pH, titratable acidity, and viable cell counts were 4.06+/-0.009-4.50+/- 0.015, 0.787+/-0.020%-1.172+/-0.018%, and 8.78+/-0.08-8.98+/- 0.06 log CFU/ml, respectively. In order to select probiotics with a high survival rate in the gut, isolates were tested to assess resistance against the artificial gastric acid and bile juice. Viable LAB counts were significantly (p<0.05) affected by the acidity. At pH 2.0, the total declines in the initial bacterial counts were 4.52+/-0.07 log for S. thermophilus St-Body-1, >7.98+/-0.03 log for E. flavescens DU-10, >7.95+/-0.05 log for E. faecium DU-12, and 3.15+/- 0.06 log for L. amylovorus DU-21. Among the strains, L. amylovorus DU-21 was the only strain that had bile tolerance under simulated gastrointestinal conditions. In order to improve EPS production by L. amylovorus DU- 21, the influence of carbon source was studied. When glucose was used as a carbon source, EPS production dramatically increased to 17.19+/-0.28 g/l (p<0.05). The maximum cell growth (10.012+/-0.012 log CFU/ml) and EPS production (18.71+/-0.19 g/l) were achieved when 15 g/ l of glucose was employed as the carbon source.     

Implementation of a multiple biomonitoring approach to evaluate the potential for impact from an industrial discharge

Over a 2-year period, an industrial discharger implemented a program to determine if there was a potential for in-stream impact from its discharge, and, if necessary, to eliminate that potential. Six basic study designs were used. These included: (1) ambient toxicity tests using indicator organisms; (2) in-stream waste concentration (IWC) chronic testing using indicator organisms; (3) on-site flow-through toxicity testing using indicator and resident species with receiving stream water as the diluent; (4) in situ acute toxicity studies using indicator and resident species; (5) biological surveys of the receiving stream; and (6) artificial stream studies. The outcome of the studies resulted in conclusive data on which to base the design of a diffuser to dilute the effluent 1:20. This concentration was well below the lowest acute no-observed-effect concentration (10% effluent) determined using sensitive resident test species. In this manner, impact from the effluent on the James River had been reduced so that even the most sensitive resident species were protected. As a result of the study, the facility's permit was modified so that toxicity tests were made only on effluent diluted with receiving stream water to represent dilution at 1Q10 rather than 100 percent effluent. Follow-up studies have concentrated on a series of toxicity tests which were designed to identify the toxicants in the final effluent.     

Use of a newly developed rapid microbial ATP bioluminescence assay to detect microbial contamination on poultry carcasses

A newly developed rapid microbial ATP bioluminescence test (R-mATP) was shown to be an adequate means to assay the microbial load of poultry carcasses. This assay utilizes differential extraction and filtration to separate somatic from microbial ATP in a very rapid timeframe. The assay requires approximately 5 min to complete; approximately 3.5 min to sample and 90 s analytical time. Correlation coefficient (r) between aerobic colony counts and R-mATP test results (n=329) was 0.82. Post-test probabilities to correctly classify carcasses with different levels of microbial contamination were as high as 98% for samples of ≥3.5 log aerobic CFU per ml. Given the rapidity of this assay, the R-mATP holds potential for monitoring the microbial load of carcasses at poultry-processing critical control points. Other potential applications of this new version of the microbial ATP bioluminescence test are discussed.     

Rapid and sensitive assay for the phytotoxin rhizobitoxine

Rhizobitoxine is a phytotoxin synthesized by some strains of the legume symbiont genus Bradyrhizobium and the plant pathogen Pseudomonas andropogonis. We demonstrate here a new enzymatic assay which is 100-fold more sensitive than previous assays and can detect as little as 1.0 pmol of rhizobitoxine. The assay is based on the inhibition of Salmonella typhimurium beta-cystathionase by rhizobitoxine. Interestingly, beta-cystathionase from Bradyrhizobium japonicum is insensitive to rhizobitoxine at concentrations lower than 75 microM.