We analyzed ABC transporter solute-binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) or two one-dimensional (1-D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2-D gel separation profiles of submembrane fractions solubilized by 1% n-dodecyl-beta-D-maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn(++), glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry (MS) is a highly accurate and sensitive means of identifying proteins. We have developed a novel method for digesting proteins on polyvinylidene difluoride (PVDF) membranes for subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis. After Tricine sodium dodecyl sulfate (SDS)-PAGE, separated proteins were electroblotted onto PVDF membranes in a semidry discontinuous buffer system, visualized by staining with Coomassie Blue, excised, digested with trypsin or lysC in 80% acetonitrile, and then analyzed by MALDI-TOF MS. This method has several advantages over in-gel digestion in terms of sample handling, sensitivity, and time. We identified 105 fmol of Bacillus subtilis SecA and 100 approximately 500 fmol of standard proteins. We also analyzed the submembrane protein fraction solubilized by 1% n-dodecyl-beta-D-maltoside from B. subtilis membranes after separation by 2-D PAGE, and identified 116 protein spots. This method can detect proteins at the 10 approximately 50 fmol level by pooling more than ten identical electroblotted protein spots.

Summary Matrix-acidizing models have traditionally underpredicted acid-stimulation benefits because of underprediction of wormhole penetration and the corresponding magnitude of completion-skin factors in vertical wells. For long horizontal wells drilled in carbonate reservoirs, productivity enhancement is a function of acid placement and effective wormhole penetration. However, prediction of wormhole penetration requires more effective analysis than that provided by current industry models. This paper presents results of matrix-acid modeling work for horizontal wells and describes a practical engineering tool for analyzing the progress of matrix-acid stimulation in carbonate reservoirs. The wormhole-growth model is based on the Buijse and Glasbergen empirical correlation. Combining with the mechanistic model of the wormhole propagation based on acid transport and fluid loss from a single wormhole, a modified Buijse-Glasbergen wormhole-growth model is developed that relates the wormhole growth rate to the in-situ injection velocity at the tip of the dominant wormhole. The wormhole constitutive model developed in this study also accounts for core-size dependencies seen in laboratory acid-flood experiments. A semianalytical flow correlation is derived for estimating interstitial velocities at the tip of the dominant wormholes based on a number of 3D FEM simulation analyses, accounting for more realistic flow regimes (radial and spherical flow) typically observed in field application. The scaleup procedure developed in this study extends the wormhole geometry and penetration from laboratory flow tests on small cores to field-sized treatments. The scaleup procedure developed in this work can be applied to cemented and uncemented horizontal wells, including barefoot and perforation-cluster completions typically employed in carbonate reservoirs. Application of this modeling shows that acid wormholing through carbonate formations can provide significant stimulation, resulting in post-stimulation skins as low as–3.5 to–4.0 vs. previously predicted values in the –1.0 to–2.0 range.

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