Biotechnology for Biofuels

This peer-reviewed article was published immediately upon acceptance. It can be downloaded, printed and distributed freely for any purposes (see copyright notice below). which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Mary E H Mandels, who spearheaded the US Army's national bioconversion studies for four decades and was an early proponent of conversion of waste biomass to readily bioconvertible sugars for the production of chemicals and transportation fuels such as ethanol, died 17 February 2008 at Natick, MA, USA. She was 90. Initially assigned to assess microbial deterioration of Army materials, her studies crisscrossed fundamental study of the cellulase enzyme from diverse microbial strains, to enzyme structure, to synergism between hydrolase components, and to large-scale enzyme production. Underpinning her studies was the need for a facile yet meaningful cellulase assay that took into consideration the insoluble and variable degree of crystallinity of the substrate, its changing nature during assay, and additionally the synergistic interaction of the multiple enzymes during hydrolysis. With her filter paper assay she mastered this broad requirement early on, as recognised in Citation Classics [1] (330 citations make it this journal's most cited paper in 1988). Trained in botany (BS, 1939) and plant physiology [2] with minors in


Commentary
Dr Mandels spent her lifelong research career at the US Army Natick Research Laboratory (NLABS), just west of Boston, MA, USA (Figure 1a, b, c). Initially assigned to assess microbial deterioration of Army materials, her studies crisscrossed fundamental study of the cellulase enzyme from diverse microbial strains, to enzyme structure, to synergism between hydrolase components, and to largescale enzyme production. Underpinning her studies was the need for a facile yet meaningful cellulase assay that took into consideration the insoluble and variable degree of crystallinity of the substrate, its changing nature during assay, and additionally the synergistic interaction of the multiple enzymes during hydrolysis. With her filter paper assay she mastered this broad requirement early on, as recognised in Citation Classics [1] (330 citations make it this journal's most cited paper in 1988).
Trained in botany (BS, 1939) and plant physiology [2] with minors in biochemistry and microbiology at Cornell University, NY, USA, Dr Mandels' background complemented the array of projects in the Pioneering Research Laboratories at NLABS. The cellulase enzyme complex was of keen military interest, it being a prime cause of the microbial deterioration of fibre and fabric: tents, cordage and clothing. She helped clarify the multicomponent nature of the cellulase complex. In the Food Science Laboratory she addressed the production of unconventional foods such as microbial single cell protein (SCP) and laboratory plant cell vegetable culture in consideration of military operation in hostile environments (for example, Antarctica), and then with the world energy crunch of the mid 1970s developed the US Army Natick recycling program centred around conversion of waste cellulosics to sugar to be fermented to ethanol, an alternative transportation fuel (gasohol = 10% ethanol) or other energyrelated chemicals. Mary, as she was known by colleagues and friends, was the eldest of five children, born to Sherman Gray and Mary Bolger Hickox at Middletown Springs, VT, USA on 12 September 1917. The family soon moved to Waterbury, CT, USA. Growing up in the Great Depression, Mary was a leader to her three sisters Corinne, Alice and Eleanor and brother Sherman, and they remained close throughout their lives. Her father was appreciative of her educational goals, yet though she had won acceptance to Cornell he still had to be nudged by Mary's science teacher to give her Images from Mary Mandels' career the support to attend college. Subsequently her mother died while she was a freshman, and then he insisted that Mary continue with her studies rather than help at home with the raising of her siblings.  (Figure 1i).) was noteworthy in producing a secreted cellulase with the outstanding quality to degrade native crystalline cellulose. Mary's role was a fundamental study in helping clarify the multicomponent nature of the cellulase enzyme complex. The synergistic action between the exosplitting and endosplitting components became evident, though arguments ensued as to the degree of synergism in that their perfect purification was somewhat dubious due to the lack of resolution by starch block electrophoresis used in that era. Only later with the subsequent cloning of the individual genes were such issues finally resolved. Induction of an enzyme in order to attack an insoluble substrate was an intriguing concept, made all the more so when in the middle of their research program T. reesei stopped producing cellulase. With the help of Fred Parrish, the cause was tracked to a change in the manufacture of glucose from starch, the hydrolysis changing from use of acid to use of amylases. Acid hydrolysis produced reversion products including the β-1,2-glucose dimer, sophorose, which turned out to be a highly active cellulase inducer of T. reesei. Thus initially commercial 'glucose', which happened to contain 0.006% sophorose, was apparently an inducer of cellulase. The new enzymatically-derived and more refined glucose could not be used in media for cellulase production [4]. The finding that sophorose was an inducer was key in subsequent studies, while tracking its presence in old commercial glucose samples is a now classic scientific 'detective' story. Dr Mandels also addressed means to inhibit cellulases, uncovering a series of natural plant inhibitors that later were shown to be of significance in the regulation of plant growth.
Transferred to the Food Science Laboratory (1962), Dr Mandels' role was to address production of foods for the military in 'hostile' environments, the latter ranging from Antarctica to the battlefield to extended space missions. The preparation of 'in house' unconventional foods was addressed through consideration of plant culture (beans, lettuce and carrots) and single cell protein (SCP) in fermentors, both exciting topics at that time. The latter included consideration of cellulolytic microbes.
Mary was reassigned to the Bioengineering, Science and Advanced Technology Laboratory in 1971, and with Elwyn Reese retiring in 1972 she went on to head this group (Figure 1d). With the onset of the oil embargo of the 1970s came the search for further energy resources. Mary spearheaded the US Army Natick Program for the enzymatic conversion of agricultural and wood wastes to sugar, to be fermented to chemicals including ethanol, to be used as alternate liquid transportation fuels. Thus developed the renowned Natick Bioenergy program under her aegis (Figure 1j). The program was all inclusive. Enhanced production of cellulase was achieved by the development of hypercellulase-producing mutants, optimisation of culture media through use of surfactants and inducers, and enhanced production was further gained by use of fed-batch and two stage continuous fermentation that included scale-up to 400 litre fermentors and 250 litre hydrolysis reactors (Figure 1e). Enzyme reuse was addressed. Numerous municipal and agricultural wastes were pretreated and evaluated with regard to their susceptibility to hydrolysis. The optimised cellulases produced up to 10% glucose solutions and attempts were made to couple such production to yeast fermentation.
The importance of the 'wastes to transportation fuel' approach was widely recognised by the press, bringing national fame to the US Army Natick program (Figure 1g, l). Mary, with Leo Spano and John Nystrom, were key advisors at the US Congressional Hearings in 1974 on alternate energy resources. The Natick Program was well received, and did not receive the classic but derogatory William Proxmire 'Golden Fleece' Award for waste of government money (Figure 1f). The Natick Laboratory had become one of the world's cellulase centres [8,9]. Such international fame attracted a series of international workers to Natick for training and discussion (Figure 1k). In like manner, Mary's expertise was widely sought and she lectured and presented short courses in China, Finland, Guatemala, India, Japan, South Africa and Mexico. Though officially retiring in 1984 she remained active in the laboratory until 1994 (Figures 2 & 3). Mary was a leader of national bioenergy studies.  There are legendary stories from the Natick program. The 'waste' substrates included both old shredded money and hydropulped confidential government documents (several tons per day). In like manner, the front pages of 50 of the top newspapers were processed (ball milled and enzymatically hydrolysed to glucose) to sugar, the comment being that the Army made 'the front page news totally digestible'. Other legends need to be put to rest. Thus reporters wanted to say that the original mould, Trichoderma sp., was isolated from a dead rotting soldier whereas it was in fact from a rotting cotton shelter half (bivouac) from Bougainville Island in the Solomon Islands [8].