Optimisation of contained Nicotiana tabacum cultivation for the production of recombinant protein pharmaceuticals

Nicotiana tabacum is emerging as a crop of choice for production of recombinant protein pharmaceuticals. Although there is significant commercial expertise in tobacco farming, different cultivation practices are likely to be needed when the objective is to optimise protein expression, yield and extraction, rather than the traditional focus on biomass and alkaloid production. Moreover, pharmaceutical transgenic tobacco plants are likely to be grown initially within a controlled environment, the parameters for which have yet to be established. Here, the growth characteristics and functional recombinant protein yields for two separate transgenic tobacco plant lines were investigated. The impacts of temperature, day-length, compost nitrogen content, radiation and plant density were examined. Temperature was the only environmental variable to affect IgG concentration in the plants, with higher yields observed in plants grown at lower temperature. In contrast, temperature, supplementary radiation and plant density all affected the total soluble protein yield in the same plants. Transgenic plants expressing a second recombinant protein (cyanovirin-N) responded differently to IgG transgenic plants to elevated temperature, with an increase in cyanovirin-N concentration, although the effect of the environmental variables on total soluble protein yields was the same as the IgG plants. Planting density and radiation levels were important factors affecting variability of the two recombinant protein yields in transgenic plants. Phenotypic differences were observed between the two transgenic plant lines and non-transformed N. tabacum, but the effect of different growing conditions was consistent between the three lines. Temperature, day length, radiation intensity and planting density all had a significant impact on biomass production. Taken together, the data suggest that recombinant protein yield is not affected substantially by environmental factors other than growth temperature. Overall productivity is therefore correlated to biomass production, although other factors such as purification burden, extractability protein stability and quality also need to be considered in the optimal design of cultivation conditions.     

Integration of microalgae cultivation with industrial waste remediation for biofuel and bioenergy production: opportunities and limitations

There is currently a renewed interest in developing microalgae as a source of renewable energy and fuel. Microalgae hold great potential as a source of biomass for the production of energy and fungible liquid transportation fuels. However, the technologies required for large-scale cultivation, processing, and conversion of microalgal biomass to energy products are underdeveloped. Microalgae offer several advantages over traditional 'first-generation' biofuels crops like corn: these include superior biomass productivity, the ability to grow on poor-quality land unsuitable for agriculture, and the potential for sustainable growth by extracting macro- and micronutrients from wastewater and industrial flue-stack emissions. Integrating microalgal cultivation with municipal wastewater treatment and industrial CO(2) emissions from coal-fired power plants is a potential strategy to produce large quantities of biomass, and represents an opportunity to develop, test, and optimize the necessary technologies to make microalgal biofuels more cost-effective and efficient. However, many constraints on the eventual deployment of this technology must be taken into consideration and mitigating strategies developed before large scale microalgal cultivation can become a reality. As a strategy for CO(2) biomitigation from industrial point source emitters, microalgal cultivation can be limited by the availability of land, light, and other nutrients like N and P. Effective removal of N and P from municipal wastewater is limited by the processing capacity of available microalgal cultivation systems. Strategies to mitigate against the constraints are discussed.     

EFFECTS OF SALINITY,NITROGEN, AND POPULATION DENSITY ON THE SURVIVAL,GROWTH, AND REPRODUCTION OF ATRIPLEX TRIANGULARIS (CHENOPODIACEAE)

Populations of Atriplex triangularis were grown under laboratory conditions in a growth chamber and manipulated in an inland Ohio saline pond in order to examine the relative effects of salinity, nitrogen fertilization, and population density on growth, reproduction, and survival. For laboratory plants, nitrogen fertilization was the most important variable, with biomass and reproductive effort being greatest at the high nitrogen level. As salinity increased, biomass decreased only in plants not limited by nitrogen. Increasing density caused biomass per plant to decrease at both high and low nitrogen levels. For field plants, density was the most important variable, with biomass per plant and survival both decreasing as density increased. As density increased, size inequality among individuals increased but biomass per unit area and individual reproductive effort remained relatively constant. Nitrogen fertilization slightly enhanced survival, but did not affect biomass. It is suggested that density-dependent processes may be significant even in relatively harsh physical environments.     

Photoautotrophic cultivating options of freshwater green microalgal Chlorococcum humicola for biomass and carotenoid production

Photoautotrophic cultivation of Chlorococcum humicola was performed in batch and continuous modes in different cultivating system arrangements to compare biomass and carotenoids’ concentration and their productivities. Batch result from stirred tank and airlift photobioreactors indicated the positive effect of increasing light intensity on growth and carotenoid production, whereas the finding from continuous cultivation indicated that carotenoid enhancement preferred high light intensity and nitrogen-deficient environment. The highest biomass (1.31?±?0.04?g?L^?1) and carotenoid (4.59?±?0.06?mg?L^?1) concentration as well as the highest productivities, 0.46?g?L^?1 d^?1 for biomass and 1.61?mg?L^?1 d^?1 for carotenoids, were obtained when maintaining high light intensity of 10 klx, BG-11 medium and 2% (v/v) CO₂ simultaneously, while the highest carotenoid content (4.84?mg?g^?1) was associated with high light intensity and nitrogen-deficient environment, which was induced by feed-modified BG-11 growth medium containing nitrate 20 folds lower than the original medium. Finally, the cultivating system arranged into smaller stirred tank photobioreactors in series yielded approximately 2.5 folds increase in both biomass and carotenoid productivities relative to using single airlift photobioreactor with equivalent working volume and similar operating condition.     

Size symmetry of competition alters biomass-density relationships

As crowded populations of plants develop, the growth of some plants is accompanied by the death of others, a process called density-dependent mortality or 'self-thinning'. During the course of density-dependent mortality, the relationship between total population biomass (B) and surviving plant density (N) is allometric: B = aN(b). Essentially, increasing population biomass can be achieved only through decreasing population density. Variation in the allometric coefficient a among species has been recognized for many years and is important for management, assessment of productivity and carbon budgets, but the causes of this variation have not been elucidated. Individual-based models predict that size-dependent competition causes variation in the allometric coefficient. Using transgenic Arabidopsis with decreased plasticity, we provide experimental evidence that morphological plasticity of wild-type populations decreases the size asymmetry of competition for light and thereby decreases density-dependent mortality. This decrease in density-dependent mortality results in more biomass at a given density under size-symmetric compared with size-asymmetric competition.     

A screening model to predict microalgae biomass growth in photobioreactors and raceway ponds

A microalgae biomass growth model was developed for screening novel strains for their potential to exhibit high biomass productivities under nutrient‐replete conditions in photobioreactors or outdoor ponds. Growth is modeled by first estimating the light attenuation by biomass according to Beer‐Lambert's Law, and then calculating the specific growth rate in discretized culture volume slices that receive declining light intensities due to attenuation. The model uses only two physical and two species‐specific biological input parameters, all of which are relatively easy to determine: incident light intensity, culture depth, as well as the biomass light absorption coefficient and the specific growth rate as a function of light intensity. Roux bottle culture experiments were performed with Nannochloropsis salina at constant temperature (23°C) at six different incident light intensities (10, 25, 50, 100, 250, and 850 µmol/m² s) to determine both the specific growth rate under non‐shading conditions and the biomass light absorption coefficient as a function of light intensity. The model was successful in predicting the biomass growth rate in these Roux bottle batch cultures during the light‐limited linear phase at different incident light intensities. Model predictions were moderately sensitive to minor variations in the values of input parameters. The model was also successful in predicting the growth performance of Chlorella sp. cultured in LED‐lighted 800 L raceway ponds operated in batch mode at constant temperature (30°C) and constant light intensity (1,650 µmol/m² s). Measurements of oxygen concentrations as a function of time demonstrated that following exposure to darkness, it takes at least 5 s for cells to initiate dark respiration. As a result, biomass loss due to dark respiration in the aphotic zone of a culture is unlikely to occur in highly mixed small‐scale photobioreactors where cells move rapidly in and out of the light. By contrast, as supported also by the growth model, biomass loss due to dark respiration occurs in the dark zones of the relatively less well‐mixed pond cultures. In addition to screening novel microalgae strains for high biomass productivities, the model can also be used for optimizing the pond design and operation. Additional research is needed to validate the biomass growth model for other microalgae species and for the more realistic case of fluctuating temperatures and light intensities observed in outdoor pond cultures. Biotechnol. Bioeng. 2013; 110: 1583–1594. © 2012 Wiley Periodicals, Inc.     

Wasserlinsen als Nutzpflanzen

Duckweeds as crop plants Members of the plant family Lemnaceae (duckweeds) are not only interesting because they represent the smallest flowering plants; they possess also the fastest rates of producing biomass. As aquatic plants, duckweed production is not in competition with other agricultural crops that require fertile land while the cultivation of duckweeds does not contribute to further eutrophication of surface water. Instead, they can be cultivated on municipal or agricultural waste water and remove the nutrients during their propagation and growth. Duckweeds can thus be used for cleaning of waste water and the resulting biomass can be valuable starting material for animal feeds and the production of biofuels. Research focusing on these goals has begun to transfer from research laboratories to pilot plants in different parts of the world, e.g. in New Jersey and North Carolina, USA; Chengdu, P. R. China; and Armidale, Australia.     

Enhancing biomass and fatty acid productivity of <Emphasis Type="Italic">Tetraselmis</Emphasis> sp. in bubble column photobioreactors by modifying light quality using light filters

Some artificial light sources able to emit photons at specific wavelengths, such as LEDs, are useful for studying the effects of light quality on microalgal growth and production of fatty acids; however, they should not be used for outdoor cultivation of microalgae to produce bioenergy. Instead, various light filters capable of selectively transmitting red, blue, and red+blue light regions in solar radiation were used to cover 0.4 L bubble column photobioreactors to cultivate Tetraselmis sp. KCTC12236BP and investigate the influence of light quality on microalgal growth and fatty acid production. Biomass and fatty acid productivities in red light (0.10 ± 0.05 g/L/day and 11.8 ± 0.5 mg/L/day, respectively) were 7 ~ 53% and 9 ~ 61% higher than other colored lights based on the same number of supplied photons, respectively. The composition of fatty acids did not change significantly in response to transmitted light qualities of the filter. The ratio of saturated to unsaturated fatty acids was 3:7, and their contents were 12% in all groups, which corresponds with the results of LEDs. Plotting biomass and fatty acid productivity over the red photon fraction in supplied light revealed that increased productivities were closely correlated with red photon fraction in the filtered light. Overall, the results presented herein indicate that enhanced production of algal fatty acid could be achieved by application of light filters in outdoor settings without artificial lights.     

Design Process of an Area-Efficient Photobioreactor

This article describes the design process of the Green Solar Collector (GSC), an area-efficient photobioreactor for the outdoor cultivation of microalgae. The overall goal has been to design a system in which all incident sunlight on the area covered by the reactor is delivered to the algae at such intensities that the light energy can be efficiently used for biomass formation. A statement of goals is formulated and constraints are specified to which the GSC needs to comply. Specifications are generated for a prototype which form and function achieve the stated goals and satisfy the specified constraints. This results in a design in which sunlight is captured into vertical plastic light guides. Sunlight reflects internally in the guide and eventually scatters out of the light guide into flat-panel photobioreactor compartments. Sunlight is focused on top of the light guides by dual-axis positioning of linear Fresnel lenses. The shape and material of the light guide is such that light is maintained in the guides when surrounded by air. The bottom part of a light guide is sandblasted to obtain a more uniform distribution of light inside the bioreactor compartment and is triangular shaped to ensure the efflux of all light out of the guide. Dimensions of the guide are such that light enters the flat-panel photobioreactor compartment at intensities that can be efficiently used by the biomass present. The integration of light capturing, transportation, distribution and usage is such that high biomass productivities per area can be achieved.     

Growth and hare, Lepus timidus, resistance of white birch, Betula pendula, clones grown in different soil types

Many plant species have the ability to expand laterally through space by clonal growth. Plant pathogens can affect clonal growth characteristics thereby altering the success of host plants within populations and of clonal species within communities. We conducted a greenhouse experiment to determine the effects of the systemic fungal pathogen, Epichloë glyceriae , on clonal growth patterns of its host grass, Glyceria striata . We found that infected and uninfected plants produced similar total biomass and numbers of tillers plus primary stolons per mother ramet. However, biomass allocation to tillering (vegetative growth) vs stolon production (clonal growth) was significantly affected by pathogen infection. Infected plants produced more stolons and clonal growth biomass than uninfected plants while mother ramets of uninfected plants produced more tillers and biomass than infected plants. Stolon architecture of infected and uninfected plants also differed. In two of three populations, infected plants produced stolons with greater biomass and shorter spacer lengths, even though mean stolon lengths were similar for infected and uninfected plants. These results contrast strongly with most other clonal plant-pathogen systems where infected plants are less vigorous and have reduced clonal growth compared to uninfected plants. Greater clonal growth may be an effective mechanism for host genotypes to persist and spread when seed production is prevented, as is the case with castrating pathogens like Epichloë glyceriae .     

籽粒苋R104生长发育与环境条件关系的研究

本文对籽粒苋(Amaranthus cruentus)生长发育与气温、光照、水、肥和土壤含盐量等因子的关系,进行了试验研究,为在生长季节较短的地区(当地无霜期只有100天左右)推广应用于生产提供理论依据和技术措施。R104是一种喜温植物,在日平均温度达到10℃左右,才能出苗,当日平均温上升到20—23℃时,生长最为迅速,当日平均温下降到5℃左右时,生长处于停滞状态。它也是一种喜光的C4植物,在土壤水分不亏缺条件下,光照上升到14万Lx,亦不呈现萎蔫状态,在遮荫条件下,植株生长细弱矮小。适宜的土壤水分和肥力是发挥籽粒苋高产优势的重要条件,在气候温暖、阳光充足的生长季节,适时进行灌溉和施肥,能使它的产量成倍增加,每公顷青饲产量可达100t左右的高产。R104还具有较强的耐盐碱能力,在土壤pH值 8.5,含盐量0.6%以下,仍能正常生长发育,土壤含盐量达到0.8%,生长发育受到影响,产量明显下降。     

Conceptual design of LED-based hydroponic photobioreactor for high-density plant cultivation

A novel hydroponic photobioreactor is proposed for high-density cultivation of plants. This cultivation can be achieved by growing plants on a floatable platform, allowing the roots to directly contact a continuously aerated nutrient solution. Plant growth of Mentha x piperita (peppermint) can be shown to strongly correlate with the light intensity at incident light intensities between 0 and 650 &mgr;mol m(-)(2) s(-)(1). For a constant incident light intensity (I(0) = 420 &mgr;mol m(-)(2) s(-)(1)), the overall specific growth rates of these plants are found to be strongly dependent on the plant density. They range from 0.023 to 0.075 d(-)(1) for plants grown at a density range from 16 to 256 plants m(-)(2). A simple mathematical model is presented that allows one to predict these effects of light intensity and plant density on peppermint growth. Light delivery is derived from the modification of Beer-Lambert's law. From this, the relationship between the light extinction coefficient and plant density can be experimentally determined. The light transport can then be coupled with plant growth kinetics under light-limiting conditions. The predicted growth results agree reasonably well with most experimental results from a growth period of 17-20 days. On the basis of these simulation results, we suggest that a more efficient way of delivering light to this photobioreactor can be attained by supplying light from both the top and the bottom of the plant shoots. The proposed design takes advantage of the small size and low weight of light emitting diodes, which allow them to be mounted on platforms for delivering light closer to the plants.     

Serial optimization of biomass production using microalga Nannochloris oculata and corresponding lipid biosynthesis

As energy and environment have become urgent issues, there has been increasing needs to develop alternative energy source, such as microalgal bio-fuel. In this study, we investigated the growth and lipid contents of microalgae Nannochloris oculata under various environmental conditions for biodiesel production. Our results indicated that biomass productivities of N. oculata were correlated with increasing nitrogen concentrations up to 37.5 ppm. High irradiance using 230-250 μmol/m(2) led to higher biomass yields than low irradiance of 160-180 μmol/m(2). Biomass productivities increased further by manipulating surface to volume ratio (S/V), which in turn enhanced light penetration. Finally, optimal biomass productivities (1.04 g/l day) could be achieved by the supplementation of yeast extract. Lipid contents and fatty acid profiles of N. oculata were affected by the different growth conditions. Lipid contents of N. oculata decreased as nitrogen concentration increased. Lower temperature (15 °C) resulted in higher lipid content than higher temperature (25 °C). Fatty acid profiles of N. oculata indicated that palmitic acid (C16:0) and linoleic acid (C18:2) were the two most abundant fatty acids, but the supplementation of yeast extract increased linolenic acid (C18:3) content. Our results suggested the feasibility of N. oculata for the biodiesel production.     

Trickle-bed root culture bioreactor design and scale-up: growth, fluid-dynamics, and oxygen mass transfer

Trickle-bed root culture reactors are shown to achieve tissue concentrations as high as 36 g DW/L (752 g FW/L) at a scale of 14 L. Root growth rate in a 1.6-L reactor configuration with improved operational conditions is shown to be indistinguishable from the laboratory-scale benchmark, the shaker flask (mu=0.33 day(-1)). These results demonstrate that trickle-bed reactor systems can sustain tissue concentrations, growth rates and volumetric biomass productivities substantially higher than other reported bioreactor configurations. Mass transfer and fluid dynamics are characterized in trickle-bed root reactors to identify appropriate operating conditions and scale-up criteria. Root tissue respiration goes through a minimum with increasing liquid flow, which is qualitatively consistent with traditional trickle-bed performance. However, liquid hold-up is much higher than traditional trickle-beds and alternative correlations based on liquid hold-up per unit tissue mass are required to account for large changes in biomass volume fraction. Bioreactor characterization is sufficient to carry out preliminary design calculations that indicate scale-up feasibility to at least 10,000 liters.     

Cultivation of the microalga <Emphasis Type="Italic">Neochloris oleoabundans</Emphasis> for biofuels production and other industrial applications (a review)

Microalgae cultivation for biofuels production and other applications has gained considerable interest recently. Despite their simple structures, microalgae can accumulate significant amounts of neutral lipids per dry cell weight compared to other energy crops. Neochloris oleoabundans is a promising microalga known for its high lipid content and biomass growth rate compared to other species cultivated for biofuels synthesis; therefore, it is considered as a suitable candidate for biodiesel synthesis. This review paper covers several key aspects associated with the cultivation and applications of the microalga N. oleoabundans. Biomass composition, factors affecting the growth, and biomass and lipid productivities of this species were addressed. In addition, different growth conditions as well as alternative readily available nutrient media to support the growth of N. oleoabundans were presented in this review.     

Fluctuations in continuous mammalian cell bioreactors with retention.

Continuous flow bioreactors with cell retention have been increasingly used for the cultivation of mammalian cells. The potential advantages of such bioreactors are high cell concentrations and volumetric productivities. In many reported cases, these systems have shown fluctuations in cell concentrations of various frequency and magnitude. To analyze the dynamics of the fluctuations, a model-based approach is followed. Simulations showed that large fluctuations in biomass resulted in response to fluctuations in the retention ratio when the system is operated at high dilution rate and high cell retention. The dependence of cell concentration fluctuations on variations in dilution rate and retention ratio was established by a cross-correlation statistical analysis on available experimental data. The slower dynamics and the fluctuation propensity of retention systems suggest that continuous culture without retention is more convenient for kinetic studies. In all likelihood, continuous culture with retention can be stabilized by controlling both the retention ratio and the dilution rate.     

On the relative efficiency of two- vs. one-stage production of astaxanthin by the green alga Haematococcus pluvialis

Haematococcus pluvialis under stress conditions overproduces the valuable red ketocarotenoid astaxanthin. Two proposed strategies for commercial production are under current analysis. One separates in time the production of biomass (optimal growth, green stage) and pigment (permanent stress, red stage), while the other uses an approach based on continuous culture under limiting stress at steady state. The productivities, efficiencies and yields for the pigment accumulation in each case have been compared and analyzed in terms of the algal basic physiology. The two-stage system indoors yields a richer astaxanthin product (4% of dry biomass) with a final astaxanthin productivity of 11.5 mg L(-1) day(-1), is more readily upscalable and amenable to outdoors production. Furthermore, each stage can be optimized for green biomass growth and red pigment accumulation by adjusting independently the respective ratio of effective irradiance to cell density. We conclude that the two-stage system performs better (by a factor of 2.5-5) than the one-stage system, and the former is best fit in an efficient mass production setup.     

The relationship of diversity and biomass in phytoplankton communities weakens when accounting for species proportions

The relationship between productivity (or biomass) and species diversity in ecological communities remains a hotly debated topic. While much is already known about vascular plants, little is known in other types of organisms. We used a broad and standardized database of phytoplankton samples from the Czech Republic, containing 413 samples of various types of stagnant waters to evaluate this relationship. Biomass was characterized by the total biovolume/ml, the total number of individuals/ml and cells/ml all giving similar results. All these indicators spanned over five orders of magnitude while the number of species ranged between 1 and 57. Diversity was characterized by indices of Hill’s unified notation series progressively accounting for species proportion effects. The number of species showed an asymmetric unimodal relationship with biomass. The relationship weakened when considering diversity indices including species proportions. At very low productivity values (characterized by low biomass), diversity was probably restricted by the ability of algae and cyanobacteria to survive a lack of nutrients, in high productivities, by the competition for light. Medium productivities, where maximum diversity was found, exhibited large variability of diversity values (including very low ones), suggesting that low diversity of phytoplankton samples can be caused by multitude of factors.     

伞形科两种植物幼苗生长对光照强度的可塑性响应

将明党参（Changium smyrnioides Wolff）和峨参（Anthriscus sylvestris Hoffm．）幼苗置于按全光照百分率为100％（S100）、65％（S65）和25％（S25）的人工控制光环境下处理。结果表明,光照强度显著影响明党参和峨参幼苗的生长：明党参在S6,生长最好,株高、冠幅、叶长、叶宽、地上地下生物量等均达到最大值,且S65的明党参生物量是S100的2倍、S25的3倍左右;而峨参地上部生长随着光照强度的减弱而增强,S25生长最好,但地下部生长则在S65生长最好,表现出与明党参同样的响应规律。明党参在不同光照强度下生长速率差异显著,S65显著高于其余两个处理;峨参虽然也在S65生长速率最大,但在不同光照强度间无差异。明党参生长速率明显小于峨参,总生物量约为峨参的1／3—1／8,相对于峨参而言明党参是一个在幼苗阶段生长缓慢的物种。     

Optimizing microalgal production in raceway systems

The industrial exploitation of microalgae is characterized by the production of high‐value compounds. Optimization of the performance of microalgae culture systems is essential to render the process economically viable. For raceway systems, the optimization based on optimal control theory is rather challenging, because the process is by essence periodically forced and, as a consequence, optimization must be carried out in a periodic framework. In this article, we propose a simple operational criterion for raceway systems that when integrated in a strategy of closed‐loop control allows attaining biomass productivities very near to the theoretical maximal productivities. The strategy developed was tested numerically using a mathematical model of microalgae growth in raceways. The model takes into account the temporal variation of the environmental variables temperature and light intensity and their influence on microalgae growth. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 543–552, 2013