S U M M A R YClaviceps fusiformis usually produces a stable viscous glucan during submerged culture fermentations. A new strain 139/2/1 G of the fungus, which subsequently autolysed this glucan to glucose, was studied and the autolysis ascribed to a constitutive PI -+ 3 glucanase and a P-glucosidase which were detected as soon as the fungal hyphae differentiated to a sclerotial form. The glucanase production followed a sigmoid pattern, reaching a maximum within 12 days, and the liberated glucose contributed to renewed growth towards the end of the fermentation. A sucrase and a maltase were also detected. Maximum glucan autolysis was achieved by using a large spore inoculum. This maintained minimal viscocity throughout the fermentation and, by maintaining adequate aeration, has since facilitated ergot alkaloid production by the organism. I N T R O D U C T I O NDavis, Rhodes & Shulke (1965) described the significant decrease of extracellular glucan in submerged cultures of Plectania occidentalis and a Helotium sp. towards the end of the fermentation and they presumed that this was due to the secretion of an ' exocellular glucanase ' by the organism. However, no autolysis was observed during studies on the structurally similar glucan produced by Claviceps fusiformis (Buck, Chen, Dickerson, & Chain, I 968). The production of alkaloids in submerged cultures by C. fusiformis is adversely affected by the formation of the viscous glucan, and during investigations on the alkaloid fermentation process a new variant strain (I 39/2/1 G ) arose spontaneously and was selected for its ability to autolyse the extracellular. glucan. The usual non-autolysing strains yielded a stable and highly viscous growth within 7 days, whereas the new variant autolysed completely after a further 7 to 10 days. In view of the structure of the extracellular glucan, its autolysis by strain 139/2/1 G has been investigated to determine whether this is due to the activity of glucanases similar to those reported by Reese & Mandels (1959) for a variety of fungi. sucrose + asparagine liquid medium was sterilized in 500 ml. Erlenmeyer flasks at 106" for 15 min. A modified medium with 10% mannitol (w/v) instead of the sucrose was also used. METHODS OriginSeed cultures. A spore suspension (2 to 6 x 109 spores/ml.) was prepared from 14-day agar cultures and I ml. inoculated into each 500 ml. flask. Usually the fermentation was completed without further transfer, but in some experiments the first (seed) stage flasks were subsequently used as a source of inoculum for a second stage. Where mycelial inocula were used an aqueous suspension was prepared by crushing the sparsely sporing mycelium from 3-day agar cultures.Flasks were incubated at 27' on a rotary shaker (Buck et al. 1968). For sampling, duplicate flasks were removed at intervals from the shaker and the course of fermentation was followed by the following parameters : microscopic examination and measurement of pH value, sugar utilization, nitrogen utilization, dry wejght of mycelium and polysaccharid...

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A strain of Claviceps fusiformis produced clavine alkaloid (principally agroclavine) in yields of 4 mglml within 6 days when grown in a sucrose-ammonium sulphate-inorganic salts medium in 400 I stirred fermenters. Alkaloid accumulated rapidly during the growth phase, most of the synthesis coinciding with a mycelial growth form consisting of plectenchymatic hyphae, the cells of which resembled the constituent cells of naturally occurring C. fusiformis sclerotia. The broth was rendered low in glucan by the activity of a ,8-glucanase and this, together with increased fermenter shaft speed, ensured that oxygen supply was not limiting throughout the fermentation. Alkaloid was extracted efficiently from the glucanfree culture filtrate by a solvent extraction procedure involving sequential transfer of the product into n-butanol, aqueous tartaric acid and chloroform, followed by crystallization from acetone. Improved alkaloid yield (6 mg/ml) was obtained by using a modified medium containing increased concentrations of magnesium sulphate, zinc sulphate and potassium dihydrogen orthophosphate, in a 400 1 multi-stage fermentation.

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