Journal of General Microbiology (1983), 129, 251-253. Printed in Great Britain 25 1 SHORT COMMUNICATION Straw as a Substrate for Cooperative Nitrogen Fixation By J. M. LYNCH* AND S. H. T. HARPER Agricultural Research Council Letcombe Laboratory, Wantage, Uxon OX12 9JT, U. K. (Received 14 September 1982) Inoculation of wheat straw, contained in glass columns and moistened by continuous recirculation of a solution containing mineral salts, with a cellulolytic fungus, Penicillium corylophilum, and a N2-fixing anaerobe, Clostridium butyricurn, increased the decomposition rate constant from 0.0096 d-l to 0.0139 d-l compared with non-inoculated straw. N2 fixation during the utilization of the straw resulted in a gain of 1 1.5 mg N (g straw lost)-* over a period of 8 weeks at 25 "C. INTRODUCTION Nitrogenase function consumes large amounts of ATP and therefore the fixation of dinitrogen in soil is generally considered to be agriculturally significant only in symbiotic associations ; inadequate carbohydrate is available for a significant contribution from the free-living N2- fixers (Postgate & Hill, 1979). Straw is about 75% (w/w) cellulosic (Harper & Lynch, 1981 a, b) and provides a potential major source of carbon and energy to the soil microflora (Lynch & Panting, 1980), but because its presence can decrease crop yield (Lynch et al., 1980), in the United Kingdom it is usually burnt. We have investigated whether inoculation of straw with a cellulolytic fungus could provide enough simple sugars for co-inoculated non-cellulolytic N2- fixing bacteria to function in competition with other organisms already present on the straw and thereby increase the N content of the decaying material. Preliminary studies showed that when straw in columns was inoculated with soil, most of the isolates capable of growth on cellulose as carbon and energy source were Penicillium spp.; P. corylophilurn was the most common. Isolation on to N-free media failed to detect Azotobacter spp. as colonizers of straw despite the presence of the bacterium in the soil inoculum. N2-fixing isolates developed only under anaerobic conditions and were exclusively Clostridium spp. We therefore selected P. corylophilum and C. butyricum for the present studies. METHODS Fiask study. Wheat (Triticurn aestiuum) straw was chopped into 0.5 cm lengths and subsamples (1 g) weighed into 250 ml conical flasks containing 100 ml N-free mineral salts solution (Dalton & Postgate, 1969)or the mineral salts solution amended with ammonium chloride (2 g 1-l). Flasks were autoclaved at 15 lbf in-2 (104 kPa) for 15 min before inoculation with Penicillium corylophilum S1/3 and Ciostridium butyricurn NCIB 7423 [about lo6 spores or cells (g straw)-']. They were incubated for 3 weeks at 20 "C on a rotary shaker (100 r.p.m,). The straw was then harvested on filter paper (Whatman no. 1) but not washed, dried at 60°C and its chemical composition determined by proximate analysis (Harper & Lynch, 19816). Losses of straw cellulose and hemicellulose were calculated from the loss in total dry weight and change in chemical composition of the remaining straw, after making corrections for initial and final ash content. N content was determined by an automatic N analyser (model 1106, Erba Science, Swindon, Wilts., U.K.). Column study. Chopped wheat straw (50 g) was packed into glass columns (300 x 50 mm) to occupy a volume of 0-5 1. The columns were sealed with silicone rubber bungs carrying glass tubes for aeration and medium supply at the top and for drainage at the base. The straw was flooded with N-free mineral salts solution for 30 min and then 0022-1287/83/0001-0792 $02.00 0 1983 SGM