Previously, we used computer-controlled fermentation technology to improve the yield of filamentous phage produced in Escherichia coli by 10-fold (Grieco et al., Bioprocess Biosyst Eng 32:773–779, 2009). In the current study, three major fermentation parameters (temperature, dissolved oxygen [DO], and pH) were investigated using design of experiments (DOE) methodology. Response surface methodology (RSM) was employed to create a process model and determine the optimal conditions for maximal phage production. The experimental data fitted best to a quadratic model (p < 0.0001). Temperature and pH, but not DO, proved to be significant variables. The model predicted a theoretical optimal condition for maximal bacteriophage production at temperature of 28.1 °C and pH 6.9. A validation run resulted in phage production [3.49 × 1011 transducing units (TU)/mL] comparable to the predicted value (2.86 × 1011 TU/mL). This represented a 7-fold increase in phage production above that obtained without optimization, resulting in a 70-fold increase above that achieved by shake flask culture alone.

Filamentous phage such as M13 and fd consist of a circular, single-stranded DNA molecule surrounded by several different coat proteins. These phages have been used extensively as vectors in phage display where one of the phage coat proteins is genetically engineered to contain a unique peptide surface loop. Through these peptide sequences, a phage collection can be screened for individual phage that binds to different macromolecules or small organic and inorganic molecules. Here, we use computer-controlled bioreactors to produce large quantities of filamentous phage in the bacterial host Escherichia coli. By measuring phage yield and bacterial growth while changing the growth medium, pH and dissolved oxygen concentration, we found that the optimal conditions for phage yield were NZY medium with pH maintained at 7.4, the dO(2) held at 100% and agitation at 800 rpm. These computer-controlled fermentations result in a minimum of a tenfold higher filamentous phage production compared to standard shake flask conditions.

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During mineral processing, concentrates of sulfide minerals of economic interest are formed by froth flotation of fine ore particles. The method works well but recovery and selectivity can be poor for ores with complex mineralogy. There is considerable interest in methods that improve the selectivity of this process while avoiding the high costs of using flotation chemicals. Here we show the first application of phage biotechnology to the processing of economically important minerals in ore slurries. A random heptapeptide library was screened for peptide sequences that bind selectively to the minerals sphalerite (ZnS) and chalcopyrite (CuFeS2). After several rounds of enrichment, cloned phage containing the surface peptide loops KPLLMGS and QPKGPKQ bound specifically to sphalerite. Phage containing the peptide loop TPTTYKV bound to both sphalerite and chalcopyrite. By using an enzyme-linked immunosorbant assay (ELISA), the phage was characterized as strong binders compared to wild-type phage. Specificity of binding was confirmed by immunochemical visualization of phage bound to mineral particles but not to silica (a waste mineral) or pyrite. The current study focused primarily on the isolation of ZnS-specific phage that could be utilized in the separation of sphalerite from silica. At mining sites where sphalerite and chalcopyrite are not found together in natural ores, the separation of sphalerite from silica would be an appropriate enrichment step. At mining sites where sphalerite and chalcopyrite do occur together, more specific phage would be required. This bacteriophage has the potential to be used in a more selective method of mineral separation and to be the basis for advanced methods of mineral processing.

As components of electronic scrap, rare earth minerals are an interesting but little used source of raw materials that are highly important for the recycling industry. Currently, there exists no cost-efficient technology to separate rare earth minerals from an electronic scrap mixture. In this study, phage surface display has been used as a key method to develop peptides with high specificity for particular inorganic targets in electronic scrap. Lanthanum phosphate doped with cerium and terbium as part of the fluorescent phosphors of spent compact fluorescent lamps (CFL) was used as a target material of economic interest to test the suitability of the phage display method to the separation of rare earth minerals. One random pVIII phage library was screened for peptide sequences that bind specifically to the fluorescent phosphor LaPO :Ce ,Tb (LAP). The library contained at least 100 binding pVIII peptides per phage particle with a diversity of 1 × 10 different phage per library. After three rounds of enrichment, a phage clone containing the surface peptide loop RCQYPLCS was found to bind specifically to LAP. Specificity and affinity of the identified phage bound peptide was confirmed by using binding and competition assays, immunofluorescence assays, and zeta potential measurements. Binding and immunofluorescence assays identified the peptide's affinity for the fluorescent phosphor components CAT (CeMgAl O :Tb ) and BAM (BaMgAl O :Eu ). No affinity was found for other fluorescent phosphor components such as YOX (Y O :Eu ). The binding specificity of the RCQYPLCS peptide loop was improved 3-51-fold by using alanine scanning mutagenesis. The identification of peptides with high specificity and affinity for special components in the fluorescent phosphor in CFLs provides a potentially new strategic approach to rare earth recycling. Biotechnol. Bioeng. 2017;114: 1016-1024. © 2016 Wiley Periodicals, Inc.