Integrating photobiological hydrogen production with dye-metal bioremoval from simulated textile wastewater

The study reports production of hydrogen in photobioreactors with free (PBR_Fr) and immobilized (PBR_Imm) Nostoc biomass at enhanced and sustained rates. Before running the photobioreactors, effects of different immobilization matrices and cyanobacterial dose on hydrogen production were studied in batch mode. As hydrogen production in the PBRs declined spent biomass from the photobioreactors were collected and utilized further for column biosorption of highly toxic dyes (Reactive Red 198 + Crystal Violet) and metals (hexavalent chromium and bivalent cobalt) from simulated textile wastewater. Breakthrough time, adsorption capacity and exhaustion time of the biosorption column were studied. The photobioreactors with free and immobilized cyanobacterium produced hydrogen at average rates of 101 and 151 μmol/h/mg Chl a, respectively over 15 days, while the adsorption capacity of the spent biomass was up to 1.4 and 0.23 mg/g for metals and 15 and 1.75 mg/g for the dyes, respectively in continuous column mode.     

Growth engineering of Synechococcus elongatus PCC 7942 for mixotrophy under natural light conditions for improved feedstock production

Synechococcus elongatus PCC 7942 has been widely explored as cyanobacterial cell factory through genetic modifications for production of various value‐added compounds. However, successful industrial scale‐ups have not been reported for the system predominantly due to its obligate photoautotrophic metabolism and use of artificial light in photobioreactors. Hence, engineering the organism to perform mixotrophy under natural light could serve as an effective solution. Thus, we applied a genetically engineered strain of Synechococcus elongatus PCC 7942 expressing heterologous hexose transporter gene (galP) to perform mixotrophy under natural light in a temperature controlled environmental chamber (EC). We systematically studied the comparative performances of these transformants using autotrophy and mixotrophy, which showed 3.4 times increase in biomass productivity of mixotrophically grown transformants over autotrophs in EC. Chlorophyll a yield was found to have decreased in mixotrophic conditions, possibly indicating reduced dependency on light for energy metabolism. Although pigment yield decreases under mixotrophy, titer was found to have improved due to increased biomass productivity. Carotenoid analysis showed that zeaxanthin is the major carotenoid produced by the species which is essential for photoprotection. Our work thus demonstrates that mixotrophy under temperature controlled natural light can serve as the viable solution to improve biomass productivity of Synechococcus elongatus PCC 7942 and for commercial production of natural or engineered value added compounds from the system. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1182–1192, 2017     

Evaluating scaling of capillary photo-biofilm reactors for high cell density cultivation of mixed trophies artificial microbial consortia

Capillary biofilm reactors (CBRs) are attractive for growing photoautotrophic bacteria as they allow high cell-density cultivation. Here, we evaluated the CBR system's suitability to grow an artificial consortium composed of Synechocystis sp. PCC 6803 and Pseudomonas sp. VBL120. The impact of reactor material, flow rate, pH, O₂, and medium composition on biomass development and long-term biofilm stability at different reactor scales was studied. Silicone was superior over other materials like glass or PVC due to its excellent O₂ permeability. High flow rates of 520 μL min⁻¹ prevented biofilm sloughing in 1 m capillary reactors, leading to a 54% higher biomass dry weight combined with the lowest O₂ concentration inside the reactor compared to standard operating conditions. Further increase in reactor length to 5 m revealed a limitation in trace elements. Increasing trace elements by a factor of five allowed for complete surface coverage with a biomass dry weight of 36.8 g m⁻² and, thus, a successful CBR scale-up by a factor of 25. Practical application : Cyanobacteria use light energy to upgrade CO₂, thereby holding the potential for carbon-neutral production processes. One of the persisting challenges is low cell density due to light limitations and O₂ accumulation often occurring in established flat panel or tubular photobioreactors. Compared to planktonic cultures, much higher cell densities (factor 10 to 100) can be obtained in cyanobacterial biofilms. The capillary biofilm reactor (CBR) offers good growth conditions for cyanobacterial biofilms, but its applicability has been shown only on the laboratory scale. Here, a first scale-up study based on sizing up was performed, testing the feasibility of this system for large-scale applications. We demonstrate that by optimizing nutrient supply and flow conditions, the system could be enlarged by factor 25 by enhancing the length of the reactor. This reactor concept, combined with cyanobacterial biofilms and numbering up, holds the potential to be applied as a flexible, carbon-neutral production platform for value-added compounds.     

Scale‐down of continuous protein producing Saccharomyces cerevisiae cultivations using a two‐compartment system

In the biotechnological industry, economic decisions in investment are typically based on laboratory‐scale experiments. Scale‐down as a tool is therefore of high industrial importance to transfer the processes into larger production scale without loss in performance. In this study, large‐scale prolonged continuous cultivations with a heterologous protein producing Saccharomyces cerevisiae strain have been scaled‐down to a two‐compartment scale‐down reactor system. The effects of glucose, pH, and oxygen concentration gradients have been investigated by comparison with corresponding 300 mL standard continuous cultivations. It was found that substrate gradients within a limited range result in increased productivity of the heterologous protein under regulation of glycolytic TPI promoter and delay the decrease of protein and trehalose production during continuous cultivation. Based on these results, it is argued that introduction of variations in substrate concentration can be beneficial for industrial continuous cultivations. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:152–159, 2016     

Implementation and analysis of temperature control strategies for outdoor photobiological hydrogen production

For outdoor photobiological hydrogen production, the effective control of temperature in photobioreactors is a challenge. In this work, an internal cooling system for outdoor tubular photobioreactors was designed, built, and tested. The temperatures in the reactors with bacteria were consistently higher than those without bacteria, and were also strongly influenced by solar irradiation and ambient air temperature. The cooling protocol applied successfully kept the reactor temperatures below the threshold limit (38 °C) required for the bioprocess and provided a uniform distribution of temperature along the reactor tube length. The biomass growth and hydrogen production were similar in the reactors cooled co-currently and counter-currently. The biomass growth rate was 0.1 l/h, the maximum hydrogen production rate was 1.28 mol/m³/h, and the overall hydrogen yield obtained was 20 %. The change in the biomass was fitted using the logistic model while cumulative hydrogen production was fitted using the modified Gompertz equation.     

Membrane technology in microalgae cultivation and harvesting: A review

Membrane processes have long been applied in different stages of microalgae cultivation and processing. These processes include microfiltration, ultrafiltration, dialysis, forward osmosis, membrane contactors and membrane spargers. They are implemented in many combinations, both as a standalone and as a coupled system (in membrane biomass retention photobioreactors (BR-MPBRs) or membrane carbonation photobioreactors (C-MPBRs). To provide sufficient background on these applications, an overview of membrane materials and membrane processes of interest in microalgae cultivation and processing is provided in this work first. Afterwards, discussion about specific aspects of membrane applications in microbial cultivation and harvesting is provided, including membrane fouling. Many of the membrane processes were shown to be promising options in microalgae cultivation. Yet, significant process optimizations are still required when they are applied to enable microalgae biomass bulk production to become competitive as a raw material for biofuel production. Recent developments of the coupled systems (BR-MPBR and C-MPBR) bring significant promises to improve the volumetric productivity of a cultivation system and the efficiency of inorganic carbon capture, respectively.     

Simultaneous use of sodium nitrate and urea as nitrogen sources improves biomass composition of Arthrospira platensis cultivated in a tubular photobioreactor

Arthrospira platensis is widely cultivated in open ponds for industrial purposes. However, high‐protein A. platensis biomass produced in photobioreactors (PBRs) is recommended for pharmaceutical and cosmetic formulations. A. platensis was cultivated in a 3.5 L tubular airlift PBR using both sodium nitrate and urea as nitrogen sources. Sodium nitrate was added from the start of the cultivation using a batch process. Urea was supplied daily at exponentially increasing feeding rate using a fed‐batch process. The simultaneous optimization of the independent variables, namely, total quantity of sodium nitrate (m_T1) and total quantity of urea (m_T2), led to an optimal condition of m_T1 = 15.0 mmol/L and m_T2 = 7.5 mmol/L. Maximum biomass concentration (5183 ± 94 mg/L) corresponding to the highest biomass productivity (683 ± 13 mg/L/day) was obtained under such condition. The addition protocol of both nitrogen sources resulted in high productivities of protein (6.2 ± 0.4 mg/L/day) as well as chlorophyll‐a (372.2 ± 7.7 mg/L/day). Such innovative process could be applied in the large‐scale production of A. platensis using tubular PBR for novel applications.     

Interactions between Clostridium beijerinckii and Geobacter metallireducens in co‐culture fermentation with anthrahydroquinone‐2, 6‐disulfonate (AH2QDS) for enhanced biohydrogen production from xylose

To enhance biohydrogen production, Clostridium beijerinckii was co‐cultured with Geobacter metallireducens in the presence of the reduced extracellular electron shuttle anthrahydroquinone‐2, 6‐disulfonate (AH₂QDS). In the co‐culture system, increases of up to 52.3% for maximum cumulative hydrogen production, 38.4% for specific hydrogen production rate, 15.4% for substrate utilization rate, 39.0% for substrate utilization extent, and 34.8% for hydrogen molar yield in co‐culture fermentation were observed compared to a pure culture of C. beijerinckii without AH₂QDS. G. metallireducens grew in the co‐culture system, resulting in a decrease in acetate concentration under co‐culture conditions and a presumed regeneration of AH₂QDS from AQDS. These co‐culture results demonstrate metabolic crosstalk between the fermentative bacterium C. beijerinckii and the respiratory bacterium G. metallireducens and suggest a strategy for industrial biohydrogen production. Biotechnol. Bioeng. 2013; 110: 164–172. © 2012 Wiley Periodicals, Inc.     

Tailoring cyanobacterial cell factory for improved industrial properties

Photosynthetic biomanufacturing provides a promising solution for sustainable production of biofuels and biochemicals. Cyanobacteria are among the most promising microbial platforms for the construction of photosynthetic cell factories. Metabolic engineering of cyanobacteria has enabled effective photosynthetic synthesis of diverse natural or non-natural metabolites, while commercialization of photosynthetic biomanufacturing is usually restricted by process and economic feasibilities. In actual outdoor conditions, active cell growth and product synthesis is restricted to narrow light exposure windows of the day-night cycles and is threatened by diverse physical, chemical, and biological environmental stresses. For biomass harvesting and bioproduct recovery, energy and cost consuming processing and equipment is required, which further decreases the economic and environmental competitiveness of the entire process. To facilitate scaled photosynthetic biomanufacturing, lots of efforts have been made to engineer cyanobacterial cell properties required by robust & continual cultivation and convenient & efficient recovery. In this review, we specifically summarized recently reported engineering strategies on optimizing industrial properties of cyanobacterial cells. Through systematically re-editing the metabolism, morphology, mutualism interaction of cyanobacterial chassis cells, the adaptabilities and compatibilities of the cyanobacterial cell factories to the industrial process could be significantly improved. Cell growth and product synthesis of the tailored cyanobacterial cells could be expanded and maintained at night and in stressful environments, while convenient biomass harvesting could also be expected. For developing more feasible cyanobacterial photosynthetic biomanufacturing in large scale, we here propose the importance of tailoring industrial properties of cyanobacteria and outline the directions that should be exploited in the future.     

Microalgae--novel highly efficient starch producers

The freshwater alga Chlorella, a highly productive source of starch, might substitute for starch‐rich terrestrial plants in bioethanol production. The cultivation conditions necessary for maximizing starch content in Chlorella biomass, generated in outdoor scale‐up solar photobioreactors, are described. The most important factor that can affect the rate of starch synthesis, and its accumulation, is mean illumination resulting from a combination of biomass concentration and incident light intensity. While 8.5% DW of starch was attained at a mean light intensity of 215 µmol/(m² s¹), 40% of DW was synthesized at a mean light intensity 330 µmol/(m² s¹). Another important factor is the phase of the cell cycle. The content of starch was highest (45% of DW) prior to cell division, but during the course of division, its cellular level rapidly decreased to about 13% of DW in cells grown in light, or to about 4% in those kept in the dark during the division phase. To produce biomass with high starch content, it is necessary to suppress cell division events, but not to disturb synthesis of starch in the chloroplast. The addition of cycloheximide (1 mg/L), a specific inhibitor of cytoplasmic protein synthesis, and the effect of element limitation (nitrogen, sulfur, phosphorus) were tested. The majority of the experiments were carried out in laboratory‐scale photobioreactors, where culture treatments increased starch content to up to about 60% of DW in the case of cycloheximide inhibition or sulfur limitation. When the cells were limited by phosphorus or nitrogen supply, the cellular starch content increased to 55% or 38% of DW, respectively, however, after about 20 h, growth of the cultures stopped producing starch, and the content of starch again decreased. Sulfur limited and cycloheximide‐treated cells maintained a high content of starch (60% of DW) for up to 2 days. Sulfur limitation, the most appropriate treatment for scaled‐up culture of starch‐enriched biomass, was carried out in an outdoor pilot‐scale experiment. After 120 h of growth in complete mineral medium, during which time the starch content reached around 18% of DW, sulfur limitation increased the starch content to 50% of DW. Biotechnol. Bioeng. 2011; 108:766–776. © 2010 Wiley Periodicals, Inc.     

Techno‐economic evaluation of a two‐step biological process for hydrogen production

An integrated biological process for the production of hydrogen based on thermophilic and photo‐heterotrophic fermentation was evaluated from a technical and economic standpoint. Besides the two fermentation steps the process also includes pretreatment of the raw material (potato steam peels) and purification of hydrogen using amine absorption. The study aimed neither at determining the absolute cost of biohydrogen nor at an economic optimization of the production process, but rather at studying the effects of different parameters on the production costs of biohydrogen as a guideline for future improvements. The effect of the key parameters, hydrogen productivity and yield and substrate concentration in the two fermentations on the cost of the hydrogen produced was studied. The selection of the process conditions was based mainly on laboratory data. The process was simulated by use of the software Aspen Plus and the capital costs were estimated using the program Aspen Icarus Process Evaluator. The study shows that the photo‐fermentation is the main contributor to the hydrogen production cost mainly because of the cost of plastic tubing, for the photo‐fermentors, which represents 40.5% of the hydrogen production cost. The costs of the capital investment and chemicals were also notable contributors to the hydrogen production cost. Major economic improvements could be achieved by increasing the productivity of the two fermentation steps on a medium‐term to long‐term scale. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Thermophilic biohydrogen production: how far are we?

Apart from being applied as an energy carrier, hydrogen is in increasing demand as a commodity. Currently, the majority of hydrogen (H₂) is produced from fossil fuels, but from an environmental perspective, sustainable H₂ production should be considered. One of the possible ways of hydrogen production is through fermentation, in particular, at elevated temperature, i.e. thermophilic biohydrogen production. This short review recapitulates the current status in thermophilic biohydrogen production through fermentation of commercially viable substrates produced from readily available renewable resources, such as agricultural residues. The route to commercially viable biohydrogen production is a multidisciplinary enterprise. Microbiological studies have pointed out certain desirable physiological characteristics in H₂-producing microorganisms. More process-oriented research has identified best applicable reactor types and cultivation conditions. Techno-economic and life cycle analyses have identified key process bottlenecks with respect to economic feasibility and its environmental impact. The review has further identified current limitations and gaps in the knowledge, and also deliberates directions for future research and development of thermophilic biohydrogen production.     

国际生物制氢相关研究的知识图谱分析

氢气是一种理想的洁净能源。生物制氢技术具有能耗低、环保等优势,是目前国内外研究的热点。从能源和环境角度考虑,发展生物制氢技术都具有重要的意义。通过ISI Web of Knowledge网络数据库检索2000~2008年8月期间生物制氢的相关研究,利用作者共引分析方法,并绘制了知识图谱。该图谱显示出此研究领域存在两大主流学术群体：群体1,其研究焦点为光解水制氢两大类,包括藻类光合制氢和蓝细菌等光合细胞制氢;群体2,其研究聚集在厌氧发酵制氢研究方面,又分为暗发酵制氢和光发酵制氢。其中厌氧发酵制氢的研究人员比较密集,说明这方面的研究是目前该领域的重点。     

Outdoor cultivation of Chlamydomonas reinhardtii for photobiological hydrogen production

Photobiological hydrogen production by the unicellular green alga Chlamydomonas reinhardtii has been studied under laboratory conditions to a vast extend but has not been investigated under outdoor conditions yet. Because the hydrogen-producing hydrogenase is very sensitive to oxygen, the production must be performed in a two-stage process: generation of the required algal biomass under oxygenic photosynthesis, followed by hydrogen biosynthesis under anaerobic conditions. In order to design a sustainable process, cultivation and subsequent hydrogen production under cost-free sunlight was investigated in this work for the first time. First, cells were grown in closed photobioreactors under simulated outdoor conditions according to the light intensities of an idealized summer day (up to 2,000?μmol photons m^?2?s^?1) in order to achieve results independent of varying, and therefore not reproducible, weather conditions. The following outdoor experiments showed comparable growth characteristics and similar cell densities. However, the use of cells grown under outdoor, simulated outdoor, or high light conditions generally resulted in significantly lower hydrogen yields compared to the use of cells cultivated under low and continuous irradiance. In order to lower cultivation costs during the growth phase, the use of 10% CO₂ corresponding to the CO₂ content of flue gas was investigated. By supplying additional CO₂ during growth under the light profile corresponding to an idealized summer day, no significant increase of cell densities could be achieved, but the subsequent hydrogen production increased compared to hydrogen production of cells grown under atmospheric CO₂.     

Utilization of cyanobacterial biomass from water bloom for bioproduction of lactic acid

Cyanobacterial biomass obtained from water blooms was successfully utilized as a material for lactic acid production. The starch contained in the biomass could be converted to D- and L-lactic acid with 80–90% yield by Lactobacillus amylovorus, in a manner similar to that contained in laboratory-cultured cyanobacterial biomass. The starch was also available for L-lactic acid production with similar high yields by L. agilis and L. ruminis that specifically produce L-lactic acid. The lactic acid production from the cyanobacterial biomass did not require any supplements such as yeast extract which are essential for lactic acid production from reagent soluble starch, indicating that nutrients contained in the cyanobacterial biomass might be effectively used for the production instead of the supplements. The starch content of the fresh cyanobacterial biomass from water bloom was increased from 10 to 19 and 24% by cultivation in 1 and 5% CO₂ in air, respectively. Using such starch-rich biomass, the concentration of lactic acid produced was successfully increased without changes in the conversion yield. These results indicate that wastewater bloom cyanobacteria could be utilized for the production of a useful compound, lactic acid.     

Photosynthetic efficiency of Chlamydomonas reinhardtii in flashing light

Efficient light to biomass conversion in photobioreactors is crucial for economically feasible microalgae production processes. It has been suggested that photosynthesis is enhanced in short light path photobioreactors by mixing‐induced flashing light regimes. In this study, photosynthetic efficiency and growth of the green microalga Chlamydomonas reinhardtii were measured using LED light to simulate light/dark cycles ranging from 5 to 100 Hz at a light‐dark ratio of 0.1 and a flash intensity of 1000 µmol m⁻² s⁻¹. Light flashing at 100 Hz yielded the same photosynthetic efficiency and specific growth rate as cultivation under continuous illumination with the same time‐averaged light intensity (i.e., 100 µmol m⁻² s⁻¹). The efficiency and growth rate decreased with decreasing flash frequency. Even at 5 Hz flashing, the rate of linear electron transport during the flash was still 2.5 times higher than during maximal growth under continuous light, suggesting storage of reducing equivalents during the flash which are available during the dark period. In this way the dark reaction of photosynthesis can continue during the dark time of a light/dark cycle. Understanding photosynthetic growth in dynamic light regimes is crucial for model development to predict microalgal photobioreactor productivities. Biotechnol. Bioeng. 2011;108: 2905–2913. © 2011 Wiley Periodicals, Inc.     

Metabolic modeling to identify engineering targets for Komagataella phaffii: The effect of biomass composition on gene target identification

Genome‐scale metabolic models are valuable tools for the design of novel strains of industrial microorganisms, such as Komagataella phaffii (syn. Pichia pastoris). However, as is the case for many industrial microbes, there is no executable metabolic model for K. phaffiii that confirms to current standards by providing the metabolite and reactions IDs, to facilitate model extension and reuse, and gene‐reaction associations to enable identification of targets for genetic manipulation. In order to remedy this deficiency, we decided to reconstruct the genome‐scale metabolic model of K. phaffii by reconciling the extant models and performing extensive manual curation in order to construct an executable model (Kp.1.0) that conforms to current standards. We then used this model to study the effect of biomass composition on the predictive success of the model. Twelve different biomass compositions obtained from published empirical data obtained under a range of growth conditions were employed in this investigation. We found that the success of Kp1.0 in predicting both gene essentiality and growth characteristics was relatively unaffected by biomass composition. However, we found that biomass composition had a profound effect on the distribution of the fluxes involved in lipid, DNA, and steroid biosynthetic processes, cellular alcohol metabolic process, and oxidation‐reduction process. Furthermore, we investigated the effect of biomass composition on the identification of suitable target genes for strain development. The analyses revealed that around 40% of the predictions of the effect of gene overexpression or deletion changed depending on the representation of biomass composition in the model. Considering the robustness of the in silico flux distributions to the changing biomass representations enables better interpretation of experimental results, reduces the risk of wrong target identification, and so both speeds and improves the process of directed strain development.     

Specific Features of Phosphate Transformation on Rhodobacter sphaeroides Chromatophores

ATP production has been shown to take place on illumination of Rhodobacter sphaeroides chromatophores by a single light flash, i.e., in the absence of a proton gradient (which would form as a result of electron transport should a second flash occur). ATP synthesis was accompanied by H₂O₂ formation. Simultaneous formation of ATP and H₂O₂ is indicative of oxidative activation of phosphate during ATP synthesis, as in model systems with isolated chlorophyll. These data provide a theoretical background for selecting illumination parameters in laboratory and industrial photobioreactors used for cultivation of photosynthetic bacteria in biotechnological processes.     

Engineered catalytic biofilms for continuous large scale production of n‐octanol and (S)‐styrene oxide

This study evaluates the technical feasibility of biofilm‐based biotransformations at an industrial scale by theoretically designing a process employing membrane fiber modules as being used in the chemical industry and compares the respective process parameters to classical stirred‐tank studies. To our knowledge, catalytic biofilm processes for fine chemicals production have so far not been reported on a technical scale. As model reactions, we applied the previously studied asymmetric styrene epoxidation employing Pseudomonas sp. strain VLB120ΔC biofilms and the here‐described selective alkane hydroxylation. Using the non‐heme iron containing alkane hydroxylase system (AlkBGT) from P. putida Gpo1 in the recombinant P. putida PpS81 pBT10 biofilm, we were able to continuously produce 1‐octanol from octane with a maximal productivity of 1.3 g L day^?1 in a single tube micro reactor. For a possible industrial application, a cylindrical membrane fiber module packed with 84,000 polypropylene fibers is proposed. Based on the here presented calculations, 59 membrane fiber modules (of 0.9 m diameter and 2 m length) would be feasible to realize a production process of 1,000 tons/year for styrene oxide. Moreover, the product yield on carbon can at least be doubled and over 400‐fold less biomass waste would be generated compared to classical stirred‐tank reactor processes. For the octanol process, instead, further intensification in biological activity and/or surface membrane enlargement is required to reach production scale. By taking into consideration challenges such as biomass growth control and maintaining a constant biological activity, this study shows that a biofilm process at an industrial scale for the production of fine chemicals is a sustainable alternative in terms of product yield and biomass waste production. Biotechnol. Bioeng. 2013; 110: 424–436. © 2012 Wiley Periodicals, Inc.     

Aerobic batch cultivation in micro bioreactor with integrated electrochemical sensor array

Aerobic batch cultivations of Candida utilis were carried out in two micro bioreactors with a working volume of 100 μL operated in parallel. The dimensions of the micro bioreactors were similar as the wells in a 96‐well microtiter plate, to preserve compatibility with the current high‐throughput cultivation systems. Each micro bioreactor was equipped with an electrochemical sensor array for the online measurement of temperature, pH, dissolved oxygen, and viable biomass concentration. Furthermore, the CO₂ production rate was obtained from the online measurement of cumulative CO₂ production during the cultivation. The online data obtained by the sensor array and the CO₂ production measurements appeared to be very reproducible for all batch cultivations performed and were highly comparable to measurement results obtained during a similar aerobic batch cultivation carried out in a conventional 4L bench‐scale bioreactor. Although the sensor chip certainly needs further improvement on some points, this work clearly shows the applicability of electrochemical sensor arrays for the monitoring of parallel micro‐scale fermentations, e.g. using the 96‐well microtiterplate format. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010