International Journal of Hydrogen Energy 33 (2008) 273 – 278 www.elsevier.com/locate/ijhydene Reverse micelles as suitable microreactor for increased biohydrogen production Anjana Pandey a , ∗ , Ashutosh Pandey b a Nanotechnology and Molecular Biology Laboratory, Centre of Biotechnology, University of Allahabad, Allahabad 211002, India b Centre of Energy Studies, MNNIT, Allahabad 211004, India Received 18 June 2007; accepted 9 July 2007 Available online 5 September 2007 Abstract Reverse micelles have been shown to act as efficient microreactors for enzymic reactions and whole cell entrapment in organic (non-aqueous) media wherein the reactants are protected from denaturation by the surrounding organic solvent. These micelles are thermodynamically stable, micrometer sized water droplets dispersed in an organic phase by a surfactant. It has been observed that when whole cells of photosynthetic bacteria (Rhodopseudomonas sphaeroides or Rhodobacter sphaeroides 2.4.1) are entrapped inside these reverse micelles, the H 2 production enhanced from 25 to 35 folds. That is, 1.71 mmol (mg protein) -1 h -1 in case of R. sphaeroides which is 25 fold higher in benzene–sodium lauryl sulfate reverse micelles. Whereas, in case of R. sphaeroides 2.4.1 the H 2 production was increased by 35 fold within AOT–isooctane reverse micelles i.e. 11.5 mmol (mg protein) -1 h -1 . The observations indicate that the entrapment of whole cells of microbes within reverse micelles provides a novel and efficient technique to produce hydrogen by the inexhaustible biological route. The two microorganisms R. sphaeroides 2.4.1 (a photosynthetic bacteria) and Citrobacter Y19 (a facultative anaerobic bacteria) together are also entrapped within AOT–isooctane and H 2 production was measured i.e. 69 mmol (mg protein) -1 h -1 . The nitrogenase enzyme responsible for hydrogen production by R. sphaeroides/ R. sphaeroides 2.4.1 cells is oxygen sensitive, and very well protected within reverse micelles by the use of combined approach of two cells (R. sphaeroides 2.4.1 and Citrobacter Y19). In this case glucose present in the medium of Citrobacter Y19 serves double roles in enhancing the sustained production rate of hydrogen. Firstly, it quenches the free O 2 liberated as a side product of reaction catalyzed by nitrogenase, which is O 2 labile. Secondly, organic acid produced by this reaction is utilized by the Citrobacter Y19 as organic substrate in anaerobic conditions.  2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. Keywords: Rhodopseudomonas sphaeroides; Rhodobacter sphaeroides 2.4.1; Citrobacter Y19; Reverse micelles and H 2 production 1. Introduction Hydrogen is an efficient energy carrier that has high specific energy content per unit mass. Therefore, it is regarded as the ideal energy source in future. It is considered the cleanest en- ergy because the combustion product is only harmless water and not any greenhouse gas [1]. Microbial H 2 production can be either photosynthetic or non- photosynthetic. Photosynthetic H 2 production is carried out by algae [2,3] or some photosynthetic bacteria [4,5]. Fermenta- tive H 2 production generally gives a higher rate [1,6] and do ∗ Corresponding author. E-mail addresses: apandey70@yahoo.com (A. Pandey), apandey70@rediffmail.com (A. Pandey). 0360-3199/$ - see front matter  2007 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2007.07.013 not rely on the availability of light sources [7], although the H 2 conversion yield (mmol H 2 mmol -1 substrate) is lower than that with photosynthetic process. For practical exploitation of anoxygenic phototrophic bacteria, stable and prolonged hydro- gen production is essential. Anoxygenic phototrophic bacteria represent a suitable future route for hydrogen production. These bacteria are able to utilize different carbon sources. Cell immobilization techniques have been successfully used for maximum and sustained H 2 production by bacteria [8]. Entrapment of microorganisms within different reverse mi- celles has been used for photoproduction of H 2 [9]. Reverse micelles consist of three components: amphiphilic surfactant molecules, water and a non-polar organic solvent. The polar heads of the surfactant molecules are directed toward the inte- rior of a water-containing sphere, whereas the aliphatic tails are