Biotechnology Progress

Only within the past decade has the potential of metal biosorption by biomass materials been well established. For economic reasons, of particular interest are abundant biomass types generated as a waste byproduct of large‐scale industrial fermentations or certain metal‐binding algae found in large quantities in the sea. These biomass types serve as a basis for newly developed metal biosorption processes foreseen particularly as a very competitive means for the detoxification of metal‐bearing industrial effluents. The assessment of the metal‐binding capacity of some new biosorbents is discussed. Lead and cadmium, for instance, have been effectively removed from very dilute solutions by the dried biomass of some ubiquitous species of brown marine algae such as Ascophyllum and Sargassum, which accumulate more than 30% of biomass dry weight in the metal. Mycelia of the industrial steroid‐transforming fungi Rhizopus and Absidia are excellent biosorbents for lead, cadmium, copper, zinc, and uranium and also bind other heavy metals up to 25% of the biomass dry weight. Biosorption isotherm curves, derived from equilibrium batch sorption experiments, are used in the evaluation of metal uptake by different biosorbents. Further studies are focusing on the assessment of biosorbent performance in dynamic continuous‐flow sorption systems. In the course of this work, new methodologies are being developed that are aimed at mathematical modeling of biosorption systems and their effective optimization. Elucidation of mechanisms active in metal biosorption is essential for successful exploitation of the phenomenon and for regeneration of biosorbent materials in multiple reuse cycles. The complex nature of biosorbent materials makes this task particularly challenging. Discussion focuses on the composition of marine algae polysaccharide structures, which seem instrumental in metal uptake and binding. The state of the art in the field of biosorption is reviewed in this article, with many references to recent reviews and key individual contributions.

The application of biotechnology to the production of commodity products (fuels, chemicals, and materials) offering benefits in terms of sustainable resource supply and environmental quality is an emergent area of intellectual endeavor and industrial practice with great promise. Such “biocommodity engineering” is distinct from biotechnology motivated by health care at multiple levels, including economic driving forces, the importance of feedstocks and cost‐motivated process engineering, and the scale of application. Plant biomass represents both the dominant foreseeable source of feedstocks for biotechnological processes as well as the only foreseeable sustainable source of organic fuels, chemicals, and materials. A variety of forms of biomass, notably many cellulosic feedstocks, are potentially available at a large scale and are cost‐competitive with low‐cost petroleum whether considered on a mass or energy basis, and in terms of price defined on a purchase or net basis for both current and projected mature technology, and on a transfer basis for mature technology. Thus the central, and we believe surmountable, impediment to more widespread application of biocommodity engineering is the general absence of low‐cost processing technology. Technological and research challenges associated with converting plant biomass into commodity products are considered relative to overcoming the recalcitrance of cellulosic biomass (converting cellulosic biomass into reactive intermediates) and product diversification (converting reactive intermediates into useful products). Advances are needed in pretreatment technology to make cellulosic materials accessible to enzymatic hydrolysis, with increased attention to the fundamental chemistry operative in pretreatment processes likely to accelerate progress. Important biotechnological challenges related to the utilization of cellulosic biomass include developing cellulase enzymes and microorganisms to produce them, fermentation of xylose and other nonglucose sugars, and “consolidated bioprocessing” in which cellulase production, cellulose hydrolysis, and fermentation of soluble carbohydrates to desired products occur in a single process step. With respect to product diversification, a distinction is made between replacement of a fossil resource‐derived chemical with a biomass‐derived chemical of identical composition and substitution of a biomass‐derived chemical with equivalent functional characteristics but distinct composition. The substitution strategy involves larger transition issues but is seen as more promising in the long term. Metabolic engineering pursuant to the production of biocommodity products requires host organisms with properties such as the ability to use low‐cost substrates, high product yield, competitive fitness, and robustness in industrial environments. In many cases, it is likely to be more successful to engineer a desired pathway into an organism having useful industrial properties rather than trying to engineer such often multi‐gene properties into host organisms that do not have them naturally. Identification of host organisms with useful industrial properties and development of genetic systems for these organisms is a research challenge distinctive to biocommodity engineering. Chemical catalysis and separations technologies have important roles to play in downstream processing of biocommodity products and involve a distinctive set of challenges relative to petrochemical processing. At its current nascent state of development, the definition and advancement of the biocommodity field can benefit from integration at multiple levels. These include technical issues associated with integrating unit operations with each other, integrating production of individual products into a multi‐product biorefinery, and integrating biorefineries into the broader resource, economic, and environmental systems in which they function. We anticipate that coproduction of multiple products, for example, production of fuels, chemicals, power, and/or feed, is likely to be essential for economic viability. Lifecycle analysis is necessary to verify the sustainability and environmental quality benefits of a particular biocommodity product or process. We see biocommodity engineering as a legitimate focus for graduate study, which is responsive to an established personnel demand in an industry that is expected to grow in the future. Graduate study in biocommodity engineering is supported by a distinctive blend of intellectual elements, including biotechnology, process engineering, and resource and environmental systems.

Electron microscopy of lignocellulosic biomass following high‐temperature pretreatment revealed the presence of spherical formations on the surface of the residual biomass. The hypothesis that these droplet formations are composed of lignins and possible lignin carbohydrate complexes is being explored. Experiments were conducted to better understand the formation of these “lignin” droplets and the possible implications they might have on the enzymatic saccharification of pretreated biomass. It was demonstrated that these droplets are produced from corn stover during pretreatment under neutral and acidic pH at and above 130 °C, and that they can deposit back onto the surface of residual biomass. The deposition of droplets produced under certain pretreatment conditions (acidic pH; T > 150 °C) and captured onto pure cellulose was shown to have a negative effect (5–20%) on the enzymatic saccharification of this substrate. It was noted that droplet density (per unit area) was greater and droplet size more variable under conditions where the greatest impact on enzymatic cellulose conversion was observed. These results indicate that this phenomenon has the potential to adversely affect the efficiency of enzymatic conversion in a lignocellulosic biorefinery.

Maltodextrins of varying molecular weights, maltose, and sucrose were used to study the effect of molecular weight, water plasticization, and composition on glass transition temperature (T_g). All maltodextrins were plasticized by water, and the decrease of T_g was linear with water activity over the range of 0.11–0.85. The plasticization effect of water was similar for maltodextrins having various molecular weights. Effects of molecular weight and composition on the T_g of maltodextrins could be correlated by using relationships reported previously for polymers. The quantitative results obtained can be applied to formulate food and related materials having desired processability and storage stability.

N‐Glycans at Asn₂₉₇ in the Fc domain of IgG molecules are required for Fc receptor‐mediated effector functions such as antibody‐dependent cell‐mediated cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC). In this study we have specifically remodeled the Fc N‐glycans of intact recombinant IgG₁ therapeutic monoclonal antibody (Mab) products, Rituxan and Herceptin, with a soluble recombinant rat β‐1,4‐N‐acetylglucosaminyltransferase III (rGnTIII) produced by baculovirus‐infected insect cells. N‐Glycan remodeling in vitro permitted a controlled and selective transfer of a bisecting β1,4‐linked GlcNAc to the core β‐linked mannose of degalactosylated Mab N‐glycans to yield Mabs varying in bisecting GlcNAc content from 31% to 85%. This was confirmed by analysis of N‐glycans by both normal phase HPLC and MALDI‐MS, the latter yielding the expected mass increase of 203.2 Da with no other oligosaccharide modifications evident. ADCC of remodeled Rituxan and Herceptin Mabs was determined using peripheral blood mononuclear cells as effectors and either CD20⁺ (SKW6.4 and SU‐DHL‐4) or Her2⁺ (SKBR‐3) target cells, respectively. A conserved 10‐fold increase in ADCC was observed for both remodeled therapeutic Mabs with high (>80%) bisecting GlcNAc content. In contrast, although the presence of a bisecting GlcNAc had minimal effect on CDC, degalactosylation of Rituxan reduced CDC by approximately half, relative to unmodified (variably galactosylated) control Mab. In summary, our data suggests that in vitro remodeling of therapeutic Mab Fc N‐glycans may be utilized to control the therapeutic efficacy of Mabs in vivo and to offer a more “humanized” glycoform profile for recombinant Mab products.

Bioethanol is a fuel‐grade ethanol made from trees, grasses, and waste materials. It represents a sustainable substitute for gasoline in today's passenger cars. Modeling and design of processes for making bioethanol are critical tools used in the U.S. Department of Energy's bioethanol research and development program. We use such analysis to guide new directions for research and to help us understand the level at which and the time when bioethanol will achieve commercial success. This paper provides an update on our latest estimates for current and projected costs of bioethanol. These estimates are the result of very sophisticated modeling and costing efforts undertaken in the program over the past few years. Bioethanol could cost anywhere from $1.16 to $1.44 per gallon, depending on the technology and the availability of low cost feedstocks for conversion to ethanol. While this cost range opens the door to fuel blending opportunities, in which ethanol can be used, for example, to improve the octane rating of gasoline, it is not currently competitive with gasoline as a bulk fuel. Research strategies and goals described in this paper have been translated into cost savings for ethanol. Our analysis of these goals shows that the cost of ethanol could drop by 40 cents per gallon over the next ten years by taking advantage of exciting new tools in biotechnology that will improve yield and performance in the conversion process.

Differential scanning calorimetry was used to determine the phase transitions of dried and rehumidified amorphous lactose, sucrose, and a mixture of sucrose and Amioca. Glass‐transition, crystallization, and melting temperatures decreased with increasing moisture content. The time to crystallization of amorphous lactose held isothermally above the glass‐transition temperature decreased as the temperature was increased. Isothermal crystallization time of lactose was a function of the temperature difference between the holding temperature and the glass‐transition temperature independently of moisture content. Amorphous biological materials are metastable showing temperature, moisture content, and time‐dependent phase transitions that affect their drying behavior, stickiness, storage stability, and quality.

Sepabeads‐EP (a new epoxy support) has been utilized to immobilize‐stabilize the enzyme penicillin G acylase (PGA) via multipoint covalent attachment. These supports are very robust and suitable for industrial purposes. Also, the internal geometry of the support is composed by cylindrical pores surrounded by the convex surfaces (this offers a good geometrical congruence for reaction with the enzyme), and it has a very high superficial density of epoxy groups (around 100 μmol/mL). These features should permit a very intense enzyme‐support interaction. However, the final stability of the immobilized enzyme is strictly dependent on the immobilization protocol. By using conventional immobilization protocols (neutral pH values, nonblockage of the support) the stability of the immobilized enzyme was quite similar to that achieved using Eupergit C to immobilize the PGA. However, when using a more sophisticated three‐step immobilization/stabilization/blockage procedure, the Sepabeads derivative was hundreds‐fold more stable than Eupergit C derivatives. The protocol used was as follows: (i) the enzyme was first covalently immobilized under very mild experimental conditions (e.g., pH 7.0 and 20 °C); (ii) the already immobilized enzyme was further incubated under more drastic conditions (higher pH values, long incubation periods, etc.) in order to “facilitate” the formation of new covalent linkages between the immobilized enzyme molecule and the support; (iii) the remaining epoxy groups of the support were blocked with very hydrophilic compounds to stop any additional interaction between the enzyme and the support. This third point was found to be critical for obtaining very stable enzymes: derivatives blocked with mercaptoethanol were much less stable than derivatives blocked with glycine or other amino acids. This was attributed to the better masking of the hydrophobicity of the support by the amino acids (having two charges).