A COMPARISON OF CARBOHYDRATE LOSS AND CARBON DIOXIDE PRODUCTION DURING FERMENTATION BY BARLEY ROOTS

IN A PREVIOUS paper (Phillips, 1947), the author showed that barley seedlings and older barley plants produce considerably more CO2 than alcohol under anaerobic conditions. The "extra" CO2 is in part due to a release of CO2 bound in bicarbonates, but the origin of much of it is unexplained. In the present study, the rate of carbohydrate loss during an anaerobic period was measured and compared with the alcohol and CO2 production in an attempt to determine the origin of the extra CO2. MATERIALS AND METHODS. Roots of barley plants 2-4 weeks old were used as experimental material. The roots were not sterile, but experiments lasted only 4 hr., and bacterial contamination did not appear to be serious. For a nutrient solution, Lake Mendota water was buffered to pH 5.0 with KH2PO4. This water is high in most mineral nutrients and supported good growth for 4 weeks. The plants were grown in pyrex crystallizing dishes 17 cm. in diameter and 9 cm. in depth, with mosquito netting fastened across the top on which the seeds were placed. The dishes were covered with a bell j ar until the roots reached the nutrient solution, then uncovered and the solution aerated vigorously through capillary tubes. After about 3 days the plants were transferred from a darkroom to the greenhouse, and aeration continued. The mat of roots weighed about 30 g. at the end of 2 weeks. Experimental procedure.-The roots were clipped just below the netting, washed well, dried gently on filter paper, weighed into 10-20 g. samples, and placed with 200 ml. of distilled water in 500 ml. wash bottles with standard taper joints. Wash bottle heads were inserted, and a stream of pure N2 was passed through the flask at a rapid rate. The N2 was freed of 02 and CO2 by the method of Peters and Van Slyke (1932). The gas from one flask was passed through an absorber containing alkali for CO2 determinations. At the end of 4 hr., the roots were removed and dried in a vacuum oven at 80 ?C. for sugar analyses, or phospho-tungstic acid was added as a killing agent and the contents of the flask distilled for alcohol analysis. Control sets of roots were killed in a similar manner at the beginning of each experiment. For experiments in the Warburg respirometer, the roots were cut into lengths of about 1 in., mixed well, dried on filter paper, and weighed into samples of proper size. Analyses.-For determination of CO2, the gas from one flask of roots was passed through a sintered glass aerator into 100 ml. of 0.05 N NaOH. The carbonate formed was measured by titration to 1Received for publication August 16, 1948. Most of the work reported in this paper was performed in the laboratories of the Department of Botany, Universitv nf Wisnsin. the phenolphthalein and methyl orange end points with 0.05 N HCI. A blank determination was performed in which N2 was bubbled through a flask containing only water, and this value was suttracted in every case. Bound CO2 was released from the tissue by treatment with 10 per cent trichloracetic acid, collected in alkali, and titrated in the same manner. Carbon dioxide production was also measured in a standard Warburg respirometer. The flasks, containing about 0.5 g. of root segments in 2 ml. of distilled water, were flushed with purified N2, closed, and placed in a water bath at 25?C. Carbon dioxide production was calculated from readings on the manometer in the usual manner. Alcohol was determined by the method of Harger (1935) on a solution obtained by distilling 150 ml. from the original flask, adding alkali, and redistilling until 100 ml. were collected. This method includes acetaldehyde in the results. Reducing sugars and sucrose were determined by the method of Hassid (1937). These are combined as "total sugars" and expressed in mM. of glucose. The material was analyzed for starch by the method of Hassid et al. (1940) but the starch content of the roots was below the limits of this method, and insignificant in comparison with the sugars.