Effect of the Nutritional Status of Semi-continuous Microalgal Cultures on the Productivity and Biochemical Composition of <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>

The rotifer Brachionus plicatilis was cultured using the microalga Isochrysis aff. galbana clone T-ISO as feed. T-ISO was cultured semi-continuously with daily renewal rates of 10%, 20%, 30%, 40%, and 50% of the volume of cultures. The increase of renewal rate led to increasing nutrient and light availability in microalgal cultures, which caused differences in the biochemical composition of microalgal biomass. Growth rate, individual dry weight, organic content, and biomass productivity of rotifer cultures increased in response to higher growth rate in T-ISO cultures. Rotifer growth rate showed a strong negative correlation (R ² = 0.90) with the C/N ratio of microalgal biomass. Rotifer dry weight was also affected by nutrient availability of T-ISO cultures, increasing up to 50% from nutrient-limited to nutrient-sufficient conditions. Consequently, biomass productivity of rotifer cultures increased more than twofold with the increase of renewal rate of T-ISO cultures. Rotifer organic content underwent the same trend of total dry weight. Maximum content of polyunsaturated fatty acids was reached in rotifers fed T-ISO from the renewal rate of 40%, with percentages of docosahexaenoic acid (22:6ω-3, DHA) and eicosapentaenoic acid (20:5ω-3, EPA) of 11% and 5% of total fatty acids, respectively. Selecting the most appropriate conditions for microalgal culture can therefore enhance the nutritive quality of microalgal biomass, resulting in a better performance of filter feeders and their nutrient content, and may constitute a useful tool to improve the rearing of fish larvae and other aquaculture organisms that require live feed in some or all the stages of their life cycle.     

Growth Rate of the Microalga Tetraselmis suecica Changes the Biochemical Composition of Artemia Species

The impact of different microalgal semicontinuous cultures on growth and biochemical composition in the next link of the food chain was tested using the filter feeder Artemia species as a model. The marine microalga Tetraselmis suecica was cultured semicontinuously with renewal rates between 10% and 50% and used to feed Artemia. Microalgal cultures maintained with a low renewal rate that had biochemical composition similar to that of the stationary-phase cultures commonly used in aquaculture produced poor growth and survival and low food-conversion efficiency compared to cultures maintained with a high renewal rate. Changes in the renewal rate in microalgal cultures also resulted in important changes in the gross biochemical composition of the filter feeder. The gross biochemical composition of the Artemia resembled that of the microalgae used as food except for total lipid content. The percentage of protein in the organic fraction of Artemia increased from 45% to 65% of the organic weight with increasing renewal rates in the microalgal cultures, while the carbohydrate percentage decreased under the same conditions. Higher renewal rates resulted in higher lipid percentages in the microalga, but in Artemia the percentage of lipids decreased from 19% of the organic weight with a renewal rate of 10%, to 13% with a renewal rate of 50%. The percentage of all polyunsaturated fatty acids in Artemia, including 20:5n-3, increased slightly with increasing renewal rates in the microalgal cultures. Results emphasize the importance of controlling microalgal nutritional value for the success of aquaculture food chains in which filter feeders are involved. Received October 15, 2000; accepted December 29, 2000.     

Analysis of Telemetric Time Series data for Periodic Components Using Dq-Fit

DQ-FIT and CV-SORT have been developed to facilitate the automatic analysis of data sampled by radiotelemetry, but they can also be used with other data sampled in chronobiological settings. After import of data, DQ-FIT performs conventional linear, as well as rhythm analysis according to user-defined specifications. Linear analysis includes calculation of mean values, load values (percentage of values above a defined limit), highest and lowest readings, and areas under the (parameter-time) curve (AUC). All of these parameters are calculated for the total sampling interval and for user-defined day and night periods. Rhythm analysis is performed by fitting of partial Fourier series with up to six harmonics. The contribution of each harmonic to the overall variation of data is tested statistically; only those components are included in the best-fit function that contribute significantly. Parameters calculated in DQ-FIT's rhythm analysis include mesor, amplitudes, and acrophases of all rhythmic components; significance and percentage rhythm of the combined best fit; maximum and minimum of the fitted curve and times of their occurrence. In addition, DQ-FIT uses the first derivative of the fitted curve (i.e., its slope) to determine the time and extent of maximal increases and decreases within the total sampling interval or user-defined intervals of interest, such as the times of lights on or off. CV-SORT can be used to create tables or graphs from groups of data sets analyzed by DQ-FIT. Graphs are created in CV-SORT by calculation of group mean profiles from individual best-fit curves rather than their curve parameters. This approach allows the user to combine data sets that differ in the number and/ or period length of harmonics included. In conclusion, DQ-FIT and CV-SORT can be helpful in the analysis of time-dependent data sampled by telemetry or other monitoring systems. The software can be obtained on request by every interested researcher. (Chronobiology International, 14(6), 561–574, 1997)     

Frequency Resonances of the Circadian Rhythm of CAM Under External Temperature Rhythms of Varied Period Lengths in Continuous Light*

Net CO₂ exchange (J_co2) of leaves of the CAM plant Kalanchoë daigremontiana Hamet et Perrier de la Bǎthie was studied in continuous light under the influence of temperature jumps of different period lengths. Each period consisted of two equal time intervals at a lower and a higher temperature, i.e. ?24 and ?29 °C respectively. At a period length of the imposed external temperature rhythm of 8 h the free running endogenous rhythm of J_co2 with a period length of 21.7 h was expressed. Under external rhythms of 12–28 h period length there was entrainment of J_co2. Under external rhythms of 40 and 56 h the time course of J_co2 became arrhythmic. In power spectra of the J_co2 time series obtained by Fourier analysis, besides the dominant frequency, there were other frequency peaks with lower Fourier amplitudes of J_co2. It was noted that these are natural number multiples of the major frequencies. A comparison shows that these frequencies correspond to the harmonic overtones of the basic oscillation of the free running endogenous rhythm of J_co2 at 24°C, viz. 1/22.5 h^?1. The overall power of the enforced oscillations shows a typical resonance behaviour around the natural, free running frequency. The intriguing observation that external temperature jump rhythms lead to resonance, not only of the basic oscillation but also of its harmonic overtones, is discussed in relation to earlier findings suggesting the occurrence of paths between order and deterministic chaos in the CAM rhythm.     

Active extracellular substances of Bacillus thuringiensis ITRI‐G1 induce microalgae self‐disruption for microalgal biofuel

Microalgal cultures are a clean and sustainable means to use solar energy for CO₂ fixation and fuel production. Microalgae grow efficiently and are rich in oil, but recovering that oil is typically expensive and consumes much energy. Therefore, effective and low‐cost techniques for microalgal disruption and oil or lipid extraction are required by the algal biofuel industry. This study introduces a novel technique that uses active extracellular substances to induce microalgal cell disruption. A bacterium indigenous to Taiwan, Bacillus thuringiensis, was used to produce the active extracellular substances, which were volatile compounds with high thermal stability. Approximately 74% of fresh microalgal cells were disrupted after a 12‐h treatment with the active extracellular substances. Algal lipid extraction efficiency was improved and the oil extraction time was decreased by approximately 37.5% compared with the control treatment. The substances effectively disrupted fresh microalgal cells but not dehydrated microalgal cells. An analysis of microalgal DNA from fresh cells after disruption treatment demonstrated typical DNA laddering, indicating that disruption may have resulted from programmed cell death. This study revealed that biological treatments are environmentally friendly methods for increasing microalgal lipid extraction efficiency, and introduced a microalgal cell self‐disruption mechanism.     

Revisiting Verhulst and Monod models: analysis of batch and fed-batch cultures

The paper re-evaluates Verhulst and Monod models. It has been claimed that standard logistic equation cannot describe the decline phase of mammalian cells in batch and fed-batch cultures and in some cases it fails to fit somatic growth data. In the present work Verhulst, population-based mechanistic growth model was revisited to describe successfully viable cell density (VCD) in exponential and decline phases of batch and fed-batch cultures of three different CHO cell lines. Verhulst model constants, K, carrying capacity (VCD/ml or μg/ml) and r, intrinsic growth factor (h⁻¹) have physical meaning and they are of biological significance. These two parameters together define the course of growth and productivity and therefore, they are valuable in optimisation of culture media, developing feeding strategies and selection of cell lines for productivity. The Verhulst growth model approach was extended to develop productivity models for batch and fed-batch cultures. All Verhulst models were validated against blind data (R² > 0.95). Critical examination of theoretical approaches concluded that Monod parameters have no physical meaning. Monod-hybrid (pseudo-mechanistic) batch models were validated against specific growth rates of respective bolus and continuous fed-batch cultures (R² ≈ 0.90). The reduced form of Monod-hybrid model C_L/(K_L + C_L) describes specific growth rate during metabolic shift (R² ≈ 0.95). Verhulst substrate-based growth models compared favourably with Monod-hybrid models. Thus, experimental evidence implies that the constants in the Monod-hybrid model may not have physical meaning but they behave similarly to the biological constants in Michaelis–Menten enzyme kinetics, the basis of the Monod growth model.     

Growth,lipid content,productivity, and fatty acid composition of tropical microalgae for scale‐up production

Biomass and lipid productivity, lipid content, and quantitative and qualitative lipid composition are critical parameters in selecting microalgal species for commercial scale‐up production. This study compares lipid content and composition, and lipid and biomass productivity during logarithmic, late logarithmic, and stationary phase of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp. grown in L1‐, f/2‐, and K‐medium. Of the tested species, Tetraselmis sp. exhibited a lipid productivity of 3.9–4.8 g m^?2 day^?1 in any media type, with comparable lipid productivity by Nannochloropsis sp. and Isochrysis sp. when grown in L1‐medium. The dry biomass productivity of Tetraselmis sp. (33.1–45.0 g m^?2 day^?1) exceeded that of the other species by a factor 2–10. Of the organisms studied, Tetraselmis sp. had the best dry biomass and/or lipid production profile in large‐scale cultures. The present study provides a practical benchmark, which allows comparison of microalgal production systems with different footprints, as well as terrestrial systems. Biotechnol. Bioeng. 2010;107: 245–257. © 2010 Wiley Periodicals, Inc.     

Energy-efficient cultivation of Chlamydomonas reinhardtii for lipid accumulation under flashing illumination conditions

Microalgal cultivation has been limited by the efficiency and costs associated with providing light energy, the most expensive and essential element needed for microalgal growth to a culture, particularly to cultures grown in a photo bioreactor (PBR). This study examined the economic benefits of using flashing illumination conditions in the context of microalgal cultivation. Chlamydomonas reinhardtii was cultivated under various conditions, including various inoculum sizes, light intensities, and durations of the light and dark periods. Our results showed that the highest microalgal growth efficiencies could be obtained using a large inoculum size under high intensity illumination accompanied by a 1:1 ratio of light and dark periods. The duration of the flashing light period was further optimized; permitting light energy savings of 62.5% of the light energy expended under continuous illumination conditions without reducing the biomass or lipid productivity. This study provides a more economical approach to cultivating C. reinhardtii via optimized flashing illumination without sacrificing microalgal growth or lipid content.     

Effect of nitrate feeding strategies on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana

Controlled nitrate feeding strategies for fed-batch cultures of microalgae were applied for the enhancement of lipid production and microalgal growth rates. In particular, in this study, the effect of nitrate feeding rates on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana were investigated using three feeding modes (i.e., pulse, continuous, and staged) and under two light variations on both lipid productivity and fatty acid compositions. Higher nitrate levels negatively affected lipid production in the study. Increasing the light intensity increased the lipid contents of the microalgae in all three fed-batch feeding modes. A maximum of 58.3% lipid- to dry weight ratio was achieved when using pulse-fed cultures at an illumination of 200 μmol photons m⁻² s⁻¹ and 10 mg/day of nitrate feeding. This condition also resulted in the maximum lipid productivity of 44.6 mg L⁻¹ day⁻¹. The fatty acid compositions of the lipids consisted predominantly of long-chain fatty acids (C:16 and C:18) and accounted for 70% of the overall fatty acid methyl esters. Pulse feeding mode was found to significantly enhance the biomass and lipid production. The other two feeding modes (continuous and staged) were not ideal for lipid and biomass production. This study demonstrates the applicability of pulse feeding strategies in fed-batch cultures as an appropriate cultivation strategy that can increase both lipid accumulation and biomass production.     

Isolation and identification of herbivorous ciliates from contaminated microalgal cultures

Ciliates are a common but understudied group of grazers that can invade microalgal cultures. To estimate the potential impact of ciliates on microalgal culture productivity, the identification of species that can invade these cultures is essential. Furthermore, isolation of these herbivorous ciliates allows to use them in experiments that investigate the impact of ciliate grazing on the productivity of microalgal cultures. The main aims of this study were to isolate and identify ciliates that invade cultures of the freshwater microalgae Chlorella and Chlamydomonas, and to establish a live collection of these ciliates for usage in future experiments. To this end, we optimized a method for isolating ciliates from contaminated microalgal cultures and we developed a new PCR primer set for amplifying the partial 18S rDNA of ciliates belonging to the classes Spirotrichea, Oligohymenophorea and Colpodea. As a result, we isolated 11 ciliates from microalgal enrichment cultures inoculated with non-sterile dust and various freshwater sources. Of these 11 species, 7 were found to be feeding on Chlamydomonas. Ciliate species that fed on Chlorella could not be isolated in this study. Ciliate species feeding on Chlamydomonas were identified based on a combination of morphological observations and molecular analyses of partial 18S rDNA sequences.     

On-line measurement of the substrate concentrations in <Emphasis Type="Italic">Pichia pastoris</Emphasis> fermentations using FT-IR/ATR

The glycerol and methanol concentrations in Pichia pastoris fermentations were measured on-line using Fourier transform infrared spectroscopy and an attenuated total reflection probe. Partial least squares regression was used to obtain calibration models. The models were regressed on synthetic multi-component spectra and semi-synthetic fermentation broth spectra. These were obtained by spectral addition. The accuracy for the on-line measurement of glycerol, given as standard error of prediction (SEP), was determined to 0.68 g/l, and the SEP of methanol was 0.13 g/l. We show how reliable calibration models are obtained relatively effortlessly by replacing extensive sampling from the reactor with simple mathematical manipulations of the model regression spectra.     

Toxicity of cadmium and zinc on two microalgae, <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> and <Emphasis Type="Italic">Desmodesmus pleiomorphus</Emphasis>, from Northern Portugal

Aquatic environments often contain toxic heavy metals that may enter the food web via uptake by microalgae and eventually cause severe poisoning problems at higher trophic levels. The effects of Cd and Zn cations upon growth of two native green microalgal species, Scenedesmus obliquus and Desmodesmus pleiomorphus (previously isolated from a polluted site in Northern Portugal), were accordingly evaluated. Growth inhibition of the microalgal cells was determined following exposure for 96 h to several initial concentrations of aqueous solutions of either of those two metals. At the higher end of Cd and Zn experimental concentration ranges, a significant reduction in cell density was observed in the cultures; EC₅₀ values, calculated after fitting a Weibull model to the experimental data, were 0.058 and 1.92 mg L⁻¹ for Cd and 16.99 and 4.87 mg L⁻¹ for Zn in the case of S. obliquus and D. pleiomorphus, respectively. One observed that S. obliquus can tolerate higher Zn concentrations than D. pleiomorphus, but the reverse holds regarding exposure to Cd.     

Genetic programming of polynomial harmonic networks using the discrete Fourier transform

This paper presents a genetic programming system that evolves polynomial harmonic networks. These are multilayer feed-forward neural networks with polynomial activation functions. The novel hybrids assume that harmonics with non-multiple frequencies may enter as inputs the activation polynomials. The harmonics with non-multiple, irregular frequencies are derived analytically using the discrete Fourier transform. The polynomial harmonic networks have tree-structured topology which makes them especially suitable for evolutionary structural search. Empirical results show that this hybrid genetic programming system outperforms an evolutionary system manipulating polynomials, the traditional Koza-style genetic programming, and the harmonic GMDH network algorithm on processing time series.     

A novel polyculture growth model of native microalgal communities to estimate biomass productivity for biofuel production

Native polyculture microalgae is a promising scheme to produce microalgal biomass as biofuel feedstock in an open raceway pond. However, predicting biomass productivity of native polycultures microalgae is incredibly complicated. Therefore, developing polyculture growth model to forecast biomass yield is indispensable for commercial-scale production. This research aims to develop a polyculture growth model for native microalgal communities in the Minamisoma algae plant and to estimate biomass and biocrude oil productivity in a semicontinuous open raceway pond. The model was built based on monoculture growth of polyculture species and it is later formulated using species growth, polyculture factor (k_value), initial concentration, light intensity, and temperature. In order to calculate species growth, a simplified Monod model was applied. In the simulation, 115 samples of the 2014–2015 field dataset were used for model training, and 70 samples of the 2017 field dataset were used for model validation. The model simulation on biomass concentration showed that the polyculture growth model with k_value had a root-mean-square error of 0.12, whereas model validation provided a better result with a root-mean-square error of 0.08. Biomass productivity forecast showed maximum productivity of 18.87 g/m²/d in June with an annual average of 13.59 g/m²/d. Biocrude oil yield forecast indicated that hydrothermal liquefaction process was more suitable with a maximum productivity of 0.59 g/m²/d compared with solvent extraction which was only 0.19 g/m²/d. With satisfactory root-mean-square errors less than 0.3, this polyculture growth model can be applied to forecast the productivity of native microalgae.     

Glucose‐6‐Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose‐6‐phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19‐fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09‐fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose‐6‐phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.     

Elevated acetyl‐CoA by amino acid recycling fuels microalgal neutral lipid accumulation in exponential growth phase for biofuel production

Microalgal neutral lipids [mainly in the form of triacylglycerols (TAGs)], feasible substrates for biofuel, are typically accumulated during the stationary growth phase. To make microalgal biofuels economically competitive with fossil fuels, generating strains that trigger TAG accumulation from the exponential growth phase is a promising biological approach. The regulatory mechanisms to trigger TAG accumulation from the exponential growth phase (TAEP) are important to be uncovered for advancing economic feasibility. Through the inhibition of pyruvate dehydrogenase kinase by sodium dichloroacetate, acetyl‐CoA level increased, resulting in TAEP in microalga Dunaliella tertiolecta. We further reported refilling of acetyl‐CoA pool through branched‐chain amino acid catabolism contributed to an overall sixfold TAEP with marginal compromise (4%) on growth in a TAG‐rich D. tertiolecta mutant from targeted screening. Herein, a three‐step α loop‐integrated metabolic model is introduced to shed lights on the neutral lipid regulatory mechanism. This article provides novel approaches to compress lipid production phase and heightens lipid productivity and photosynthetic carbon capture via enhancing acetyl‐CoA level, which would optimize renewable microalgal biofuel to fulfil the demanding fuel market.     

Song harmonic content as a parameter determining acoustic orientation behaviour in the cricketTeleogryllus oceanicus (Le Guillou)

Summary Analyses of natural songs from the cricketTeleogryllus oceanicus confirm (after Hutchings and Lewis 1984) that the songs are rich in harmonics which extend up to around 55 kHz. A series of synthetic song models with a varying harmonic content were used to determine the relevance of these harmonics to the orientation behaviour of the insects.In two-choice experiments in which song models were presented simultaneously, crickets showed a clear discrimination between a song with harmonics and a song without. Female insects orientated preferentially to a song with harmonics. Interestingly, males preferred a song model without harmonics in the two-choice situation.Two experimental regimes were used to test orientation accuracy, a forced-choice (Y maze) phonotactic experiment and a study of free phonotaxis in the behavioural arena. The results of both experimental approaches confirm that crickets orientate more accurately to a song with harmonics and can discriminate smaller angles to sound sources ahead and lateral to the insect.Presentations of song models in which the fundamental (5 kHz) was attenuated relative to the 2nd harmonic have given results that imply a process of neural integration (two-tone or side band suppression) of the fundamental and the high-frequency harmonics.Abbreviations see Methods     

Tris not only controls the pH in microalgal cultures,but also feeds bacteria

Tris (Tris(hydroxymethyl)amino methane), a compound often used as a buffer in microalgal culture media, sustains active bacterial growth in non-axenic microalgal cultures when sodium phosphate is present. The low pH levels caused by bacterial growth and probably the depletion of phosphorus in the medium caused the collapse ofPhaeodactylum tricornutum cultures resulting in a reduction of microalgal growth from 32 x 10⁶ to 1.1 x 10⁶ cells ml^–1. This emphasizes the need for care when interpreting the results of non-axenic microalgae cultures in which Tris or other organic buffer is added.