The objective of the present study was to investigate the optimal nutritional requirements for hydrogen production from carbon monoxide by biological water-gas shift (WGS) reaction with Carboxydothermus hydrogenoformans using orthogonal layout methods. Cultures of C. hydrogenoformans on the medium as formulated by the strain supplier (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) unexpectedly showed a large content in inorganic material. This was confirmed by the rather elevated levels of calcium and phosphorus contained in the grown biomass. As an excessive production of those minerals may interfere with the growth and catabolism rates as well as clog up biofilm-based reactors, it was desirable to minimize the mineral accumulation during growth, while keeping at maximal levels both the H(2) yield and the specific H(2) production rate (SHPR). PO(4)(3-), HCO(-)(3), Ca(2+) and Mg(2+) were considered as potentially the major factors of the mineral accumulation. The experiments were designed according to the Taguchi's orthogonal method, using the above factors at three levels, and considering the culture mineral content, the H(2) yield and the SHPR as optimization criteria. Optimal concentrations of PO(4)(3)-, HCO(3)(-), Ca(2+) and Mg(2+) were determined as (mM): 1.0, 5.0, 0.1 and 0.5, respectively. Under those culture conditions, Ca + P content decreased from 55.6 +/- 1.8 to 9.5 +/- 3% while the highest H(2) yield at 90.9 +/- 1.2% and SHPR at 0.85 +/- 0.06 mol H(2) g(-1) VSS d(-1) were achieved in bottle batch tests at 100% CO headspace atmosphere, neutral pH, a temperature of 70 degrees C, and an agitation of 100 rpm. Crown Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.