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

Highlights

• Soil samples from the archaeological site of Shahr-i Sokhta in Iran was used to retrieve microbes buried 4000 years ago.

• Archaeobotany studies were performed to investigate climate and human activities and to select soil samples.

• The failure of conventional methods of detecting culturable microbes led us to use specific culture conditions.

• Five sporogenic strains found are associated with food spoilage and all thrived on oat found in the archeological city.

• Strains were identified as Aspergillus flavus, A. restrictus, Penicillium crysogenum, Cladosporium sp., Bacillus sp.

Abstract

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 1.5–2 m 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 50 mg 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.

Introduction

The archaeological site of Shahr-i Sokhta (Burnt City in Persian) is located in southeastern Iran, about 57 km south of Zabol, the capital of the Sistan district. The site lies on a plateau-like fluvial terrace and the ruins of the ancient settlement and associated graveyard cover an area of about 151 ha (Biscione et al., 1977). Shahr-i Sokhta was the most ancient proto-urban settlement in Iranian Sistan and the excavations carried out by an Italian team in 1967–1978 (Tosi, 1968, Tosi, 1983, Salvatori and Vidale, 1997) and since 1997 by the Iranian expedition of ICAR (Sajjadi et al., 2003) revealed four periods of occupation, covering a time span from 3200 to 1800 BC (Salvatori and Vidale, 1997, Salvatori and Tosi, 2005, Piperno, 2007). The settlement was situated on a third millennium BC branch of the Helmand river, close to a lake into which the river flowed.

The particularly hot and dry climate and the saline crust that formed over most of the site has ensured preservation of unburnt organic remains with only slight deterioration. It has therefore been possible to collect large quantities of wood fragments, grains of food plants and seeds of wild grasses, as well as considerable charcoal. Analysis and study of these materials has made it possible to reconstruct the agriculture and the ancient ecosystems of this part of Sistan in the 3rd millennium BC (Biscione et al., 1974; Tosi 1978; Costantini, 1977, Costantini, 1979, Costantini and Costantini-Biasini, 1985; Sajjadi et al., 2003, Costantini et al., 2007, Sajjadi et al., 2008).

In recent years, there has been growing interest in changes in local climate and environment as major reasons why the settlement was abandoned (Biscione, 2010). Food shortage has been regarded as a direct effect of these changes. No attention has, however, been paid to potential health hazards associated with ancient urban/domestic pollution, although large garbage deposits have been found in several parts of the site. Palynology and carpology studies could provide useful information on agriculture and food practices and for reconstructing climate and the dietary habits of the inhabitants (Milanesi et al., 2006a, Milanesi et al., 2006b, Riehl, 2009), while preliminary palaeobotanical analysis of soil and vessel contents show abundant cereals, cucurbits and grapes (Costantini et al., 2007).

Light microscopy (Suyama et al., 1996) Transmission electron microscopy (Milanesi et al., 2006a, Milanesi et al., 2006b) and Scanning electron microscopy with energy dispersive X-ray has been used to study microorganism ultrastructure and soils prehistoric fertilization strategies buried for millennia (Nielsen and Kristiansen, 2014). This instrument is excellent for identifying elements indicative of metallurgical activities and metals used in prehistoric handicrafts (Wilson et al., 2008).

During excavations in 2007, soil was sampled to search for and identify fungal and bacterial spores. The aim of the study was to combine cultural, microscopy and genetic techniques to isolate and identify microorganisms that survived 4000 years of burial under a salty crust at a depth of more than one meter, in order to obtain additional information about food spoilage at Shahr-i Sokhta. The presence of specific microbial species in soil suggests that sudden abandonment of the city may not only have been influenced by diversion of the river but also by food deterioration.