Spoilage of oat bran by sporogenic microorganisms revived from soil buried 4000 years ago in Iranian archaeological site

The Bronze Age archaeological site of Shahr-i Sokhta (30° 39' N; 61° 24' E), located today in southeastern Iran, Sistan region, is a special archaeological deposit in which the exceptional preservation of human, plant and animal remains, due to the dry climate of the region, can provide detailed information on one of the first complex proto-urban societies. In recent years, there has been growing interest in changes in local climate and environment as major reasons why the settlement was abandoned about 4000 years ago. Food shortage has been regarded as a direct effect of these changes. No attention has been paid to the potential health hazards associated with ancient urban/domestic pollution, although large garbage deposits have been found in several parts of the site.During excavations in 2007, four soil samples were taken under aseptic conditions at a depth of 1.5-2m in a stratified deposit sealed by the floor of a small house, dated to the second half of the third millennium BC. Microbiological, palynological, carpological and microanalytical studies were performed on the four soil samples. Site C was identified as the most affected by human activity. Failure of conventional methods of detecting culturable and unculturable microbes in site C indicated the need for specific culture conditions suggested by palynological observations. Since oat seed residues were identified among the archaeobotanical material, oat bran was used as carbon and energy source to make a new medium to revive microbes. Coarsely ground oat bran was sterilized twice and soaked with minimal medium as sole carbon source. About 50mg of buried soil from site C was added to the medium in cell culture flasks with aerobic and anaerobic stoppers and incubated at 28-30°C and at 4°C.After incubation under aerobic and anaerobic conditions, five sporogenic microbes were identified by sequencing 16S rDNA and ITS rRNA regions: a sporogenic strain IRC3 identified as Bacillus sp. was the only isolate under anaerobic conditions, whereas under aerobic conditions four moulds were isolated: Aspergillus flavus IRC1, Penicillium crysogenum IRC2, Cladosporium sp. IRC4, and the psychrotroph Aspergillus restrictus IRC5. Bacillus sp., with 99.7% similarity to Bacillus subtilis, broke down oat bran, producing a gel, while Cladosporium sp., with 99.8% similarity to Cladosporium sphaerospermium, grew on oat bran by synthesizing intracellular lipids. All these microbes are known to spoil food and they are common where there is intense human activity.