Euphytica 140: 47–54, 2004. C  2004 Kluwer Academic Publishers. Printed in the Netherlands. 47 Exploitation of bacterial pectinolytic strains for improvement of hemp water retting Pectinolytic bacteria in water retting Elena Tamburini 1 , Alicia Gordillo Le ´ on 2 , Brunella Perito 2 , Mario Di Candilo 3 & Giorgio Mastromei ∗,2 1 Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e Biotecnologie Microbiche, University of Cagliari, Cittadella universitaria SS 554, 09042 Monserrato (CA), Italy; 2 Department of Animal Biology and Genetics “Leo Pardi”, University of Florence, Via Romana 17, 50125 Florence, Italy; 3 Istituto Sperimentale per le Colture Industriali, via di Corticella 133, 40129 Bologna, Italy; ( ∗ author for correspondence: e-mail: mastromei@dbag.unifi.it) Key words: cellulase, flax, hemp, polygalacturonase, water retting Summary Retting is the major limitation to an efficient production of textile hemp fibres. Traditional retting has been carried out by autochthonous bacterial community. Aerobic and anaerobic pectinolytic strains were isolated from hemp or flax sources and characterised. Anaerobic pectinolytic strains had a wide range of acid polygalacturonase (PG) activity, whereas aerobic isolates did not produce any acid PG activity, but only an alkalophylic one, suggesting they could play a minor role in the retting process, except in the early stages. Analysis of 16S rDNA sequences assigned anaerobic strains to the Clostridium genus and aerobic isolates to the Bacillus and Paenibacillus genus. C. felsineum and C. acetobutylicum were confirmed as the main anaerobic agents. Nevertheless, a high proportion of anaerobic and aerobic pectinolytic strains was assigned to C. saccharobutylicum and B. pumilus, respectively, both species never being described as involved in water retting. Anaerobic and aerobic strains with high PG activity were selected and characterized. PG activity is well correlated with the strain retting efficiency and improvement of the process was obtained by inoculating the retting water with spores of selected aerobic and anaerobic bacteria. An advisable feature of retting strains is the absence of cellulosolytic activity. An aerobic strain with no cellulosolytic activity was identified. In contrast, all the anaerobic isolates showed cellulosolytic activity. Mutagenesis was ineffective for selection of Cel-Pec + mutants. Localization of the C. felsineum L1/6 PG activity was investigated. Abbreviations: 16S rDNA: 16S rRNA gene, ARDRA: amplified ribosomal DNA restriction analysis, CBD: cellulose-binding domain, CMC: carboxymethylcellulose, CMCase: CMC-degrading, DNS: dinitrosalicylic acid, PG: polygalacturonase Introduction For textile productions, bast fibres of hemp (Cannabis sativa) stems must be released from the amorphous pectic matrix in a process known as retting. After retting, fibres are mechanically separated from other material by scutching. During the past years, retting process has been carried out by microbiological or chemical treatments. Chemical retting included treatments with acids and bases (Dujardin, 1948), surfactants, and chelators (Henriksson et al., 1998). However, this process is considered expensive, due to the high consumption of chemicals and energy, and environmental unfriendly,