Fumarate production with Rhizopus oryzae: utilising the Crabtree effect to minimise ethanol by-product formation.

BACKGROUND: The four-carbon dicarboxylic acids of the tricarboxylic acid cycle (malate, fumarate and succinate) remain promising bio-based alternatives to various precursor chemicals derived from fossil-based feed stocks. The double carbon bond in fumarate, in addition to the two terminal carboxylic groups, opens up an array of downstream reaction possibilities, where replacement options for petrochemical derived maleic anhydride are worth mentioning. To date the most promising organism for producing fumarate is Rhizopus oryzae (ATCC 20344, also referred to as Rhizopus delemar) that naturally excretes fumarate under nitrogen-limited conditions. Fumarate excretion in R. oryzae is always associated with the co-excretion of ethanol, an unwanted metabolic product from the fermentation. Attempts to eliminate ethanol production classically focus on enhanced oxygen availability within the mycelium matrix. In this study our immobilised R. oryzae process was employed to investigate and utilise the Crabtree characteristics of the organism in order to establish the limits of ethanol by-product formation under growth and non-growth conditions. RESULTS: All fermentations were performed with either nitrogen excess (growth phase) or nitrogen limitation (production phase) where medium replacements were done between the growth and the production phase. Initial experiments employed excess glucose for both growth and production, while the oxygen partial pressure was varied between a dissolved oxygen of 18.4% and 85%. Ethanol was formed during both growth and production phases and the oxygen partial pressure had zero influence on the response. Results clearly indicated that possible anaerobic zones within the mycelium were not responsible for ethanol formation, hinting that ethanol is formed under fully aerobic conditions as a metabolic overflow product. For Crabtree-positive organisms like Saccharomyces cerevisiae ethanol overflow is manipulated by controlling the glucose input to the fermentation. The same strategy was employed for R. oryzae for both growth and production fermentations. It was shown that all ethanol can be eliminated during growth for a glucose addition rate of 0.07 g L - 1 h - 1 . The production phase behaved in a similar manner, where glucose addition of 0.197 g L - 1 h - 1 resulted in fumarate production of 0.150 g L - 1 h - 1 and a yield of 0.802 g g - 1 fumarate on glucose. Further investigation into the effect of glucose addition revealed that ethanol overflow commences at a glucose addition rate of 0.395 g g - 1 h - 1 on biomass, while the maximum glucose uptake rate was established to be between 0.426 and 0.533 g g - 1 h - 1 . CONCLUSIONS: The results conclusively prove that R. oryzae is a Crabtree-positive organism and that the characteristic can be utilised to completely discard ethanol by-product formation. A state referred to as "homofumarate production" was illustrated, where all carbon input exits the cell as either fumarate or respiratory CO 2 . The highest biomass-based "homofumarate production": rate of 0.243 g g - 1 h - 1 achieved a yield of 0.802 g g - 1 on glucose, indicating the bounds for developing an ethanol free process. The control strategy employed in this study in conjunction with the uncomplicated scalability of the immobilised process provides new direction for further developing bio-fumarate production.

Improved continuous fumaric acid production with immobilised Rhizopus oryzae by implementation of a revised nitrogen control strategy.

A novel fermentation system was employed whereby the mycelial mat of Rhizopus oryzae was attached to a polypropylene tube. Batch operation was used for growth, while continuous operation was employed during the fumaric acid production phase. A clear decrease in respiration, fumaric acid (FA) and ethanol production was observed when zero nitrogen was fed in the production phase, with FA productivity decreasing from an initial 0.7 g L-1 h-1 to 0.3 g L-1 h-1 after 150 h. With the addition of 0.625 mg L-1 h-1 of urea FA productivity dropped to only 0.4 g L-1 h-1 after 150 h and 0.3 g L-1 h-1 after 400 h. Under these conditions it was observed that the ethanol production rate decreased 20 times faster compared with the FA production rate, therefore resulting in high FA yields towards the end of the fermentation (instantaneous 0.96 g g-1 and average 0.81 g g-1 after 400 h). Increasing the urea feed rate to 1.875 mg L-1 h-1 resulted in a clear increase in FA production and respiration rates. This condition also resulted in a 25% increase in biomass after 150 h, while the decline in the ethanol production rate was seven times lower than in the 0.625 mg L-1 h-1 urea fermentation, resulting in lower FA yields.

Laparoscopic urology training in South Africa.

BACKGROUND AND PURPOSE: Laparoscopic urologic surgery is not widely practiced in South Africa. After presenting a laparoscopic training course, we evaluated how effectively this training translated into clinical practice. SUBJECTS AND METHODS: Invitations to the course were sent to all South African urologists. Ten applicants attended the course, which consisted of dry and in-vivo animal surgery. Two questionnaires were sent out after the course. Questionnaire 1 (at course completion) aimed at identifying the precourse laparoscopic experience and expectations of the trainee. Questionnaire 2 (6 months postcourse) assessed how much laparoscopic surgery the participant had performed since the course. RESULTS: Seventeen percent of all South African urologists responded to the invitation. Prior to the course, 40% of trainees had performed >or=10 laparoscopic cases, 30% had performed <10 cases, and 30% had never performed laparoscopy, whereas 60% expected to be doing one or two cases a month after completing the course. Six months after the course, 60% had performed no laparoscopic cases. Of the three trainees who had never before done laparoscopic procedures, none had started to perform procedures since the course. The commonest procedures performed were varicocelectomy and diagnostic laparoscopy for nonpalpable testis. CONCLUSIONS: A hands-on laparoscopic training course to introduce laparoscopic urology into South African private urology practice has not translated into a satisfactory number of clinical cases being performed. The causes are likely multifactorial but are greatly influenced by social and economic forces. One possible solution may be to offer a mentor-based training program.

What is the place of internal urethrotomy in the treatment of urethral stricture disease?

As a treatment for male urethral stricture, internal urethrotomy (IU) has the advantages of ease, simplicity, speed and short convalescence. Various modifications of the single cold-knife incision in the 12 o'clock position have been proposed, but there are no prospective, randomized studies to prove their claims of greater efficacy. IU can be performed as an outpatient procedure using local anesthesia, with an indwelling silicone catheter for 3 days after the procedure. Complications of IU are usually minor, including infection and hemorrhage. The reported success rate of IU varies, mainly because of differences in the definition of success and the duration of follow-up. Strictures can recur, usually within 3-12 months of IU. There are several known risk factors for recurrence: a previous IU, penile and membranous strictures, long (>2 cm) and multiple strictures, untreated perioperative urinary infection and extensive periurethral spongiofibrosis. Repeated IU might be useful in patients who have a stricture recurrence more than 6 months after the initial procedure, but repeat IU offers no long-term cure after a third IU, or if a stricture recurs within 3 months of the first IU. Such patients should be offered urethroplasty. Repeated IU followed by long-term self-dilation is an alternative option for men with severe comorbidity and limited life expectancy, or those who have failed previous urethroplasty. Overall, IU has a lower success rate (+/-60%) than urethroplasty (+/-80-90%), but if used for selected strictures, the success rate of IU could approach that of urethroplasty.