Nonlinear predictive control for maximization of CO2 bio-fixation by microalgae in a photobioreactor

In the framework of environment preservation, microalgae biotechnology appears as a promising alternative for CO₂ mitigation. Advanced control strategies can be further developed to maximize biomass productivity, by maintaining these microorganisms in bioreactors at optimal operating conditions. This article proposes the implementation of Nonlinear Predictive Control combined with an on-line estimation of the biomass concentration, using dissolved carbon dioxide concentration measurements. First, optimal culture conditions are determined so that biomass productivity is maximized. To cope with the lack of on-line biomass concentration measurements, an interval observer for biomass concentration estimation is built and described. This estimator provides a stable accurate interval for the state trajectory and is further included in a nonlinear model predictive control framework that regulates the biomass concentration at its optimal value. The proposed methodology is applied to cultures of the microalgae Chlorella vulgaris in a laboratory-scale continuous photobioreactor. Performance and robustness of the proposed control strategy are assessed through experimental results.     

Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae

In vivo determination of neutral lipids with Nile red fluorescence has been used as a rapid screening method for certain types of microalgae, but has been unsuccessful in others, particularly those with thick, rigid cell walls that prevent penetration of the fluorescence dye into the cell. To solve the problem, a microwave-assisted Nile red staining method for microalgal lipid determination was developed. In a two-step staining protocol, 50 and 60 s were selected as the optimal microwave times for the pretreatment and staining process, respectively. Moreover, several calibration methods for quantitative analysis of neutral lipids in microalgae were investigated and compared with conventional gravimetric methods. Factors that affected the in vivo quantification of cellular neutral lipids were also investigated. Application of the new method for detection and quantification of neutral lipids in a number of green microalgae was demonstrated.     

Enrichment as a screening method for a high-growth-rate microalgal strain under continuous cultivation system

Microalgae are a promising feedstock for renewable biodiesel production. High productivity of biodiesel production from microalgae is directly related to growth rate as well as lipid content of cells. In the present study, an enrichment process in a continuous cultivation system was developed to screen a high-growth-rate microalga from a mixed culture of microalgal species; Chlorella vulgaris, Chlorella protothecoides, and Chlamydomonas reinhardtii were used as test organisms for our experiments. The time-dependent washout of mixed microalgal pool was executed to successfully enrich the C. reinhardtii, which exhibits the higher growth rate than C. vulgaris and C. protothecoides under turbidostat conditions within 75 h. The domination of C. reinhardtii in the mixed culture was validated by on-line monitoring of growth rate and flowcytometric analysis. For the time-efficient production of microalgal biomass, this screening process has a high potential to segregate the fast-growing microalgal strains from the pool of various uncharacterized microalgal species and random mutants.     

Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production

Global petroleum reserves are shrinking at a fast pace, increasing the demand for alternate fuels. Microalgae have the ability to grow rapidly, and synthesize and accumulate large amounts (approximately 20-50% of dry weight) of neutral lipid stored in cytosolic lipid bodies. A successful and economically viable algae based biofuel industry mainly depends on the selection of appropriate algal strains. The main focus of bioprospecting for microalgae is to identify unique high lipid producing microalgae from different habitats. Indigenous species of microalgae with high lipid yields are especially valuable in the biofuel industry. Isolation, purification and identification of natural microalgal assemblages using conventional techniques is generally time consuming. However, the recent use of micromanipulation as a rapid isolating tool allows for a higher screening throughput. The appropriate media and growth conditions are also important for successful microalgal proliferation. Environmental parameters recorded at the sampling site are necessary to optimize in vitro growth. Identification of species generally requires a combination of morphological and genetic characterization. The selected microalgal strains are grown in upscale systems such as raceway ponds or photobireactors for biomass and lipid production. This paper reviews the recent methodologies adopted for site selection, sampling, strain selection and identification, optimization of cultural conditions for superior lipid yield for biofuel production. Energy generation routes of microalgal lipids and biomass are discussed in detail.     

Microalgal industry in China: challenges and prospects

Over the past 15 years, China has become the major producer of microalgal biomass in the world. Spirulina (Arthrospira) is the largest microalgal product by tonnage and value, followed by Chlorella, Dunaliella, and Haematococcus, the four main microalgae grown commercially. China’s production is estimated at about two-thirds of global microalgae biomass of which roughly 90 % is sold for human consumption as human nutritional products (‘nutraceuticals’), with smaller markets in animal feeds mainly for marine aquaculture. Research is also ongoing in China, as in the rest of the world, for other high-value as well as commodity microalgal products, from pharmaceuticals to biofuels and CO₂ capture and utilization. This paper briefly reviews the main challenges and potential solutions for expanding commercial microalgae production in China and the markets for microalgae products. The Chinese Microalgae Industry Alliance (CMIA), a network founded by Chinese microalgae researchers and commercial enterprises, supports this industry by promoting improved safety and quality standards, and advancement of technologies that can innovate and increase the markets for microalgal products. Microalgae are a growing source of human nutritional products and could become a future source of sustainable commodities, from foods and feeds, to, possibly, fuels and fertilizers.     

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Microalgae have been shown as a potential bioresource for food, biofuel, and pharmaceutical products. During the growth phases with corresponding environmental conditions, microalgae accumulate different amounts of various metabolites. We quantified the neutral lipids accumulation and analyzed the swimming signatures (speed and trajectories) of the motile green alga, Dunaliella primolecta, during the lag–exponential–stationary growth cycle at different nutrient concentrations. We discovered significant changes in the neutral lipid content and swimming signatures of microalgae across growth phases. The timing of the maximum swimming speed coincided with the maximum neutral lipid content and both maxima occurred under nutrient stress at the stationary growth phase. Furthermore, the swimming trajectories suggested statistically significant changes in swimming modes at the stationary growth phase when the maximum intracellular neutral lipid content was observed. Our results provide the potential exploitation of microalgal swimming signatures as possible indicators of the cultivation conditions and the timing of microalgal harvest to maximize the lipid yield for biofuel production. The findings can also be implemented to explore the production of food and antibiotics from other microalgal metabolites with low energy costs.     

Direct colony PCR for rapid identification of varied microalgae from freshwater environment

Molecular identification of microalgal species is vital and involves sequencing of specific markers present in the genome, which are unique to a genus. PCR is a vital tool for identification of microalgae; the preparation of template DNA for PCR by traditional methods requires large amount of algal cells and a time-consuming process. One simple way to reduce these complications is to use the microalgal colonies directly for amplification of required DNA fragments from the genome. In this study, a simple cell-disrupting method, using autoclaved glass powder, has been used for direct colony PCR of microalgae. Four morphologically different microalgal strains were chosen from freshwater samples, and the PCR amplification reaction was evaluated with three different molecular markers (rbcL, internal transcribed spacer 2, and 18S rDNA). PCR amplification was optimized with less number of cells (0.04?×?10⁵), minimal quantity of glass powder (0.5 mg), and in the presence of Milli-Q water for internal transcribed spacer marker. The isolated strains were identified as Desmodesmus sp. JQ782747, Coelastrum proboscideum JQ898144, Chlorella sorokiniana JQ898145, and Scenedesmus sp. JQ782746 based on sequence similarity. This direct microalgal colony PCR proves to be a simple and rapid method for detection of varied microalgal species.     

Increased Growth of the Microalga Chlorella vulgaris when Coimmobilized and Cocultured in Alginate Beads with the Plant-Growth-Promoting Bacterium Azospirillum brasilense

Coimmobilization of the freshwater microalga Chlorella vulgaris and the plant-growth-promoting bacterium Azospirillum brasilense in small alginate beads resulted in a significantly increased growth of the microalga. Dry and fresh weight, total number of cells, size of the microalgal clusters (colonies) within the bead, number of microalgal cells per cluster, and the levels of microalgal pigments significantly increased. Light microscopy revealed that both microorganisms colonized the same cavities inside the beads, though the microalgae tended to concentrate in the more aerated periphery while the bacteria colonized the entire bead. The effect of indole-3-acetic acid addition to microalgal culture prior to immobilization of microorganisms in alginate beads partially imitated the effect of A. brasilense. We propose that coimmobilization of microalgae and plant-growth-promoting bacteria is an effective means of increasing microalgal populations within confined environments.     

The effect of ultrasound on the growth and viability of microalgae cells

Ultrasound has shown potential for both increasing microalgal lipid extraction yields and for the control of microalgal blooms through cell disruption. The effect of ultrasound on the viability of microalgae was investigated on the following species: Dunaliella salina, Chlamydomonas concordia and Nannochloropsis oculata. Sonication with a 20 kHz probe (0.086 W cm^?3) caused complete cell disruption of D. salina after 4 min. This microalgae species does not have a true cell wall. In the case of C. concordia which has a thin cell wall complete cell disruption under the same conditions took 16 min. Under the same conditions, there was no visible disruption of N. oculata, a species which has a thick cell wall. However spectro-fluorophotometer analysis of the sonicated suspension of N. oculata showed that although the cells were intact, the level of intracellular chlorophyll was reduced by ~10 %. This clearly indicated damage to the microalgal cell wall. After 16 min, treatment cultures of all three species remained viable. Programmed cell death (PCD) has been induced in some microalgal species to terminate algal blooms; ultrasonic application did not induce PCD in any species tested. The supernatant of sonicated D. salina and C. concordia has also been shown to be able to boost the growth of established cultures. These results provide important information concerning the uses of ultrasound in both the microalgal biofuels industry and for the control of microalgal blooms.     

Integration of biological waste conversion and wastewater treatment plants by microalgae cultivation

In this study, growth performance and lipid content of two microalgae species Neochloris oleoabundans and Chlorella vulgaris are monitored by using three different types of sludge waste feedstocks obtained from the water treatment plants located in Bedonia, Borgotaro and Fornovo (Montagna2000 Spa, Province of Parma, Italy). The sludge waste is optimized in order to achieve microalgal growth media and dispose of the sewage sludge produced at the wastewater treatment facilities. Both photoautotrophic and heterotrophic growth conditions are applied to the microalgal cultivations. The growth parameters of microalgae strains such as cell concentration, growth rate, optical density, cell biovolume, photosynthetic pigments and lipid contents are monitored. The amounts of total dried lipid biomass, obtained by the biological conversion of the wet sludge waste, are determined. Lipid production of microalgal cells grown in the medium optimized from sludge waste from the Fornovo site provides the highest amount of microalgal lipid content for N. oleoabundans and C. vulgaris photoautotrophic cultivations, while sludge waste from the Bedonia site provides for N. oleoabundans heterotrophic cultivation.     

Molecular Identification and Comparative Evaluation of Tropical Marine Microalgae for Biodiesel Production

Marine microalgae have emerged as important feedstock for liquid biofuel production. The identification of lipid-rich native microalgal species with high growth rate and optimal fatty acid profile and biodiesel properties is the most challenging step in microalgae-based biodiesel production. In this study, attempts have been made to bio-prospect the biodiesel production potential of marine and brackish water microalgal isolates from the west coast of India. A total of 14 microalgal species were isolated, identified using specific molecular markers and based on the lipid content; seven species with total lipid content above 20% of dry cell weight were selected for assessing biodiesel production potential in terms of lipid and biomass productivities, nile red fluorescence, fatty acid profile and biodiesel properties. On comparative analysis, the diatoms were proven to be promising based on the overall desirable properties for biodiesel production. The most potential strain Navicula phyllepta MACC8 with a total lipid content of 26.54 % of dry weight of biomass, the highest growth rate (0.58 day^?1) and lipid and biomass productivities of 114 and 431 mgL^?1 day^?1, respectively, was rich in fatty acids mainly of C16:0, C16:1 and C18:0 in the neutral lipid fraction, the most favoured fatty acids for ideal biodiesel properties. The biodiesel properties met the requirements of fuel quality standards based on empirical estimation. The marine diatoms hold a great promise as feedstock for large-scale biodiesel production along with valuable by-products in a biorefinery perspective, after augmenting lipid and biomass production through biochemical and genetic engineering approaches.     

Effects of bacterial communities on biofuel-producing microalgae: stimulation,inhibition and harvesting

Despite the great interest in microalgae as a potential source of biofuel to substitute for fossil fuels, little information is available on the effects of bacterial symbionts in mass algal cultivation systems. The bacterial communities associated with microalgae are a crucial factor in the process of microalgal biomass and lipid production and may stimulate or inhibit growth of biofuel-producing microalgae. In addition, we discuss here the potential use of bacteria to harvest biofuel-producing microalgae. We propose that aggregation of microalgae by bacteria to achieve >90% reductions in volume followed by centrifugation could be an economic approach for harvesting of biofuel-producing microalgae. Our aims in this review are to promote understanding of the effects of bacterial communities on microalgae and draw attention to the importance of this topic in the microalgal biofuel field.     

Rapid Characterization of Microalgae and Microalgae Mixtures Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures.     

Microalgal process-monitoring based on high-selectivity spectroscopy tools: status and future perspectives

Microalgae are well known for their ability to accumulate lipids intracellularly, which can be used for biofuels and mitigate CO₂ emissions. However, due to economic challenges, microalgae bioprocesses have maneuvered towards the simultaneous production of food, feed, fuel, and various high-value chemicals in a biorefinery concept. On-line and in-line monitoring of macromolecules such as lipids, proteins, carbohydrates, and high-value pigments will be more critical to maintain product quality and consistency for downstream processing in a biorefinery to maintain and valorize these markets. The main contribution of this review is to present current and prospective advances of on-line and in-line process analytical technology (PAT), with high-selectivity – the capability of monitoring several analytes simultaneously – in the interest of improving product quality, productivity, and process automation of a microalgal biorefinery. The high-selectivity PAT under consideration are mid-infrared (MIR), near-infrared (NIR), and Raman vibrational spectroscopies. The current review contains a critical assessment of these technologies in the context of recent advances in software and hardware in order to move microalgae production towards process automation through multivariate process control (MVPC) and software sensors trained on “big data”. The paper will also include a comprehensive overview of off-line implementations of vibrational spectroscopy in microalgal research as it pertains to spectral interpretation and process automation to aid and motivate development.     

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.     

Starch determination in Chlorella vulgaris—a comparison between acid and enzymatic methods

Different methods for estimating starch in Chlorella vulgaris were compared with the view of establishing a procedure suitable for rapid and accurate determination of starch content in this microalgal species. A close agreement was observed between methods that use perchloric acid and enzymatic methods that use α-amylase and amyloglucosidase to hydrolyze the starch of microalgae grown under different nitrogen culture conditions. Starch values obtained by these methods were significantly higher than those estimated by using hydrochloric acid as solubilizing and hydrolyzing agent. The enzymatic method (EM1) proved to be the most rapid and precise method for microalgal starch quantification. Furthermore, the evaluation of resistant starch by enzymatic methods assayed in nitrogen-sufficient and nitrogen-starved cells showed that no formation of this type of starch occurred in microalgae, meaning that this should not interfere with starch content determinations.     

Perspectives on microalgal CO2-emission mitigation systems — A review

The problem of climate change arising mainly from CO₂ emission is currently a critical environmental issue. Biofixation using microalgae has recently become an attractive approach to CO₂ capture and recycling with additional benefits of downstream utilization and applications of the resulting microalgal biomass. This review summarizes the history and strategies of microalgal mitigation of CO₂ emissions, photobioreactor systems used to cultivate microalgae for CO₂ fixation, current microalgae harvesting methods, as well as applications of valuable by-products. It is of importance to select appropriate microalgal species to achieve an efficient and economically feasible CO₂-emission mitigation process. The desired microalgae species should have a high growth rate, high CO₂ fixation ability, low contamination risk, low operation cost, be easy to harvest and rich in valuable components in their biomass.     

Biofouling in photobioreactors for marine microalgae

The economic and/or energetic feasibility of processes based on using microalgae biomass requires an efficient cultivation system. In photobioreactors (PBRs), the adhesion of microalgae to the transparent PBR surfaces leads to biofouling and reduces the solar radiation penetrating the PBR. Light reduction within the PBR decreases biomass productivity and, therefore, the photosynthetic efficiency of the cultivation system. Additionally, PBR biofouling leads to a series of further undesirable events including changes in cell pigmentation, culture degradation, and contamination by invasive microorganisms; all of which can result in the cultivation process having to be stopped. Designing PBR surfaces with proper materials, functional groups or surface coatings, to prevent microalgal adhesion is essential for solving the biofouling problem. Such a significant advance in microalgal biotechnology would enable extended operational periods at high productivity and reduce maintenance costs. In this paper, we review the few systematic studies performed so far and applied the existing thermodynamic and colloidal theories for microbial biofouling formation in order to understand microalgal adhesion on PBR surfaces and the microalgae–microalgae cell interactions. Their relationship to the physicochemical properties of the solid PBR surface, the microalgae cell surfaces, and the ionic strength of the culture medium is discussed. The suitability and the applicability of such theories are reviewed. To this end, an example of biofouling formation on a commercial glass surface is presented for the marine microalgae Nannochloropsis gaditana. It highlights the adhesion dynamics and the inaccuracies of the process and the need for further refinement of previous theories so as to apply them to flowing systems, such as is the case for PBRs used to culture microalgae.     

Constitutive and Chloroplast Targeted Expression of Acetyl-CoA Carboxylase in Oleaginous Microalgae Elevates Fatty Acid Biosynthesis

Photosynthetic microalgae are of burgeoning interest in the generation of commercial bioproducts. Microalgae accumulate high lipid content under adverse conditions, which in turn compromise their growth and hinder their commercial potential. Hence, it is necessary to engineer microalgae to mitigate elevated lipid accumulation and biomass. In this study, we identified acetyl-CoA carboxylase (ACCase) in oleaginous microalga Phaeodactylum tricornutum (PtACC2) and expressed constitutively in the chloroplast to demonstrate the potential of chloroplast engineering. Molecular characterization of transplastomic microalgae revealed that PtACC2 was integrated, transcribed and expressed successfully, and localized in the chloroplast. Enzymatic activity of ACCase was elevated by 3.3-fold, and the relative neutral lipid content increased substantially by 1.77-fold, and lipid content reached up to 40.8% of dry weight. Accordingly, the number and size of oil bodies markedly increased. Fatty acid profiling showed that the content of monounsaturated fatty acids increased, while polyunsaturated fatty acids decreased. This method provides a valuable genetic engineering toolbox for microalgal bioreactors with industrial significance.