Heterotrophic Cultures Research Articles

Enhancement of Biomass and Protein Production of Chlorella protothecoides in Heterotrophic Cultivation Using Expired Juices as Alternative Source of Nutrients for an Added-Value Biorefinery Scheme

Microalgae are among the most suitable sources of new protein ingredients and bioactive compounds, although their cost-effective production still remains a developmental bottleneck. Heterotrophic growth has advantages, such as higher productivity and non-dependence on light and CO2, but it has not yet been fully implemented. The aim of this study was to increase the biomass and protein of Chlorella protothecoides in heterotrophic conditions using expired fruit and vegetable juices (FVJ) and to compare the results to those obtained using a modified Bristol (MB) medium. The initial amounts of sugars (15 g L−1) and yeast extract (8 g L−1) led to the highest protein production (43%) and protein yield (5.2 g L−1) in trials using 1 L bottles. Bioreactor trials using FVJ resulted in a similar biomass productivity (2.94 g L−1 d−1) compared to MB (3.04 g L−1 d−1) and in enhanced polyunsaturated fatty acid content; nonetheless, a lower amount of essential amino acids (EAA) was obtained. Biomass was further processed by ultra-high-pressure homogenisation for protein concentration and residual pellet production. The protein extracts showed significant increases in EAA (11.8% and 26% increase in MB and FVJ, respectively). Residual biomass showed potential as a source of lutein and antimicrobial compounds. The results indicate that FVJ is a promising source of nutrients for the heterotrophic production of C. protothecoides, and that the extracts obtained present nutritional profiles and bioactivities of potential interest.

Metabolomic exploration of the physiological regulatory mechanism of the growth and metabolism characteristics of Chlorella vulgaris under photoautotrophic, mixotrophic, and heterotrophic cultivation conditions

Microalgae have various trophic modes such as photoautotrophy, mixotrophy, and heterotrophy, and the physiological and metabolic processes of algal cells in different trophic modes are different. To explore the mechanism underlying trophic mode-mediated regulation of the physiological metabolism of microalgae, Chlorella vulgaris 31 was cultivated under photoautotrophic, mixotrophic, and heterotrophic conditions. Mixotrophic and heterotrophic cultivation significantly promoted the growth, biomass, and lipid and saccharide biosynthesis of C. vulgaris, while higher photosynthetic pigment and protein content could be obtained under autotrophic conditions. Flow cytometry analysis revealed that light periodically and significantly regulated agal cell growth under the photoautotrophic condition, but a weakened effect was observed under mixotrophic and heterotrophic conditions. The mitochondrial structure was affected by enhanced aerobic respiration under mixotrophic and heterotrophic modes. Moreover, the accumulation of starch grains and lipid droplets was significantly higher than that in the photoautotrophic control; however, no significant changes were observed in the chloroplast and nucleus. Intracellular metabolites in mixotrophic and heterotrophic modes varied markedly, mainly involving saccharides, organic acids, fatty acids, and amino acids. Metabolic pathway analysis indicated that mixotrophy and heterotrophy could significantly enhance glycolysis, the tricarboxylic acid cycle, and the pentose phosphate pathway on the 3rd day of cultivation, while higher acetyl-CoA levels were available for fatty acid synthesis in the stationary phase. This finding clarified the mechanism of mixotrophic and heterotrophic growth of C. vulgaris for biomass and biomolecule production.

Optimization of nutrient utilization efficiency and productivity for algal cultures under light and dark cycles using genome-scale model process control

Algal cultivations are strongly influenced by light and dark cycles. In this study, genome-scale metabolic models were applied to optimize nutrient supply during alternating light and dark cycles of Chlorella vulgaris. This approach lowered the glucose requirement by 75% and nitrate requirement by 23%, respectively, while maintaining high final biomass densities that were more than 80% of glucose-fed heterotrophic culture. Furthermore, by strictly controlling glucose feeding during the alternating cycles based on model-input, yields of biomass, lutein, and fatty acids per gram of glucose were more than threefold higher with cycling compared to heterotrophic cultivation. Next, the model was incorporated into open-loop and closed-loop control systems and compared with traditional fed-batch systems. Closed-loop systems which incorporated a feed-optimizing algorithm increased biomass yield on glucose more than twofold compared to standard fed-batch cultures for cycling cultures. Finally, the performance was compared to conventional proportional-integral-derivative (PID) controllers. Both simulation and experimental results exhibited superior performance for genome-scale model process control (GMPC) compared to traditional PID systems, reducing the overall measured value and setpoint error by 80% over 8 h. Overall, this approach provides researchers with the capability to enhance nutrient utilization and productivity of cell factories systematically by combining genome-scale models and controllers into an integrated platform with superior performance to conventional fed-batch and PID methodologies.

Highly efficient co-production of fucoxanthin and eicosapentaenoic acid by heterotrophic cultivation of a newly isolated microalga Nitzschia sp. FZU62

Microalgae can accumulate various compounds simultaneously, thus co-production of high-value metabolites to improve the economic value of microalgae production is feasible. In this study, a microalga strain Nitzschia sp. FZU62 capable of co-producing fucoxanthin and eicosapentaenoic acid (EPA) was isolated and identified. Further, heterotrophic conditions were optimized to improve the co-production efficiency in flasks. Results showed that the optimal conditions for flask culture were cultivation for 84 h with an inoculum size of 0.47 g/L and a temperature of 19 °C. Besides, three fed-batch culture strategies with distinct nutrient compositions were explored to improve the heterotrophic performance of strain FZU62. Results indicated that the highest biomass, fucoxanthin, and EPA productivities were obtained by Fed-batch III, in which concentrated medium was pulse-fed to supply 60 % initial nutrients once glucose concentration dropped to about 2 g/L. Moreover, the heterotrophic performance was verified in a 5 L fermenter, resulting in excellent fucoxanthin and EPA productions of 63.03 and 398.60 mg/L, and productivities of 3.41 and 22.52 mg/L/d, respectively. These results, especially the EPA production and productivity, are superior to many literature reports. The present study provides new insights into the highly efficient co-production of fucoxanthin and EPA by marine microalgae and has guiding significance for commercial production in heterotrophic mode.

Biological and metabolic effects of the association between the microalga Galdieria sulphuraria and the fungus Penicillium citrinum

Contamination of microalgae cultures can reduce their productivity and affect the quality of biomass and valuable bioproducts. In this article, after having isolated and identified for the first time the filamentous fungus Penicillium citrinum from heterotrophic cultures of the red polyextremophilic microalga Galdieria sulphuraria, we investigated the biological and metabolic significance of this alga-fungus association. In the same medium, both organisms grow better in each other's presence than separately. Both cell density and cell size of G. sulphuraria increase in co-cultures compared to pure alga cultures. In co-cultures, despite very severe growth conditions, the load of P. citrinum increases compared to pure fungus cultures. Optical microscope images have shown physical contact between cells of P. citrinum hyphae and G. sulphuraria which, however, retain their morphology and cell wall intact. GC–MS-based metabolomics analysis of metabolites excreted in the culture medium shows that pure cultures of the fungus and alga and co-cultures of alga plus fungus can be easily differentiated based on their metabolic products. Indeed, a richer assortment of extracellular metabolites (comprising both products of primary and secondary metabolism) is a distinct feature of co-cultures compared to both pure alga and pure fungus cultures.

Dynamics of a PDE model with size-structure: Characterizing the growth and flocculation effects of unicellular algae

The commercial production of algae is of great practical significance to the reduction of environmental pollution, the development of bioenergy and nutritional food. Heterotrophic culture is one of the most important methods in commercial production of algae, and flocculation is usually used to collect algae cells in the reactor. The size of algae cells has a direct impact on the nutrient absorption and the flocculation effect of algae cells. In this paper, a size-structured PDE model considering the size of algae cells is established, which depicts the evolutionary relationship of the growth of algae cells, the nutrient absorption and flocculation effect. In the construction of the model, it is assumed that the number of newborn algae cells is determined by nutrition and the weighted total number of algal cells. The numerical observation shows that the model has complex dynamics, such as the appearances of the backward bifurcation of equilibrium states and bistability. The main theoretical results are as follows: (1) The well-posedness of the solution is obtained by using the fixed point theorem. (2) Under the condition of threshold quantity R0>1, the existence of the coexistence equilibrium state of algae is analyzed. Then, by using the theory of contraction semigroups, the sufficient condition for the local stability of the coexistence equilibrium state of algae is obtained. (3) Sufficient condition for the global stability of the algae-free equilibrium state is given.

Microalgae Commercialization Using Renewable Lignocellulose Is Economically and Environmentally Viable.

Conventional phototrophic cultivation for microalgae production suffers from low and unstable biomass productivity due to limited and unreliable light transmission outdoors. Alternatively, the use of a renewable lignocellulose-derived carbon source, cellulosic hydrolysate, offers a cost-effective and sustainable pathway to cultivate microalgae heterotrophically with high algal growth rate and terminal density. In this study, we evaluate the feasibility of cellulosic hydrolysate-mediated heterotrophic cultivation (Cel-HC) for microalgae production by performing economic and environmental comparisons with phototrophic cultivation through techno-economic analysis and life cycle assessment. We estimate a minimum selling price (MSP) of 4722 USD/t for producing high-purity microalgae through Cel-HC considering annual biomass productivity of 300 t (dry weight), which is competitive with the conventional phototrophic raceway pond system. Revenues from the lignocellulose-derived co-products, xylose and fulvic acid fertilizer, could further reduce the MSP to 2976 USD/t, highlighting the advantages of simultaneously producing high-value products and biofuels in an integrated biorefinery scheme. Further, Cel-HC exhibits lower environmental impacts, such as cumulative energy demand and greenhouse gas emissions, than phototrophic systems, revealing its potential to reduce the carbon intensity of algae-derived commodities. Our results demonstrate the economic and environmental competitiveness of heterotrophic microalgae production based on renewable bio-feedstock of lignocellulose.

MOLECULAR CHARACTERIZATION OF EXO-ELECTROGENIC BACTERIA FROM HIGH ANDEAN SOILS FOR THE PRODUCTION OF BIOELECTRICITY

The characterization of microbial populations that act in the anode compartment of the MFC allows to obtain pure cultures, the same ones that allow obtaining better electrical energy voltages. Therefore, the objective of this research was to molecularly characterize axenic cultures from an anodic Bio-film formed by exo-electrogenic bacteria from Páramo soil for its application for treating leachate from fruits and vegetables from the Riobamba wholesale market in central Ecuador through cells of microbial fuel. The novelty of this research is the finding of potential microorganisms from high Andean soils that can reduce pollutant loads from wastewater with energy recovery. For the experimentation, the Delftia acidovorans bacterium obtained from molecular identification of nine species and a Páramo soil solution was selected as inoculums for two treatments in triplicate. Simple configuration cells of 510 mL volume were built and data acquisition system was used to automatically collect voltage values on a computer for 15 cycles. The statistical analysis determined that there are no significant differences in the production of voltage and removal of pollutants in both treatments, obtaining similar percentages of removal of organic matter measured in BOD5, COD and N-NH4. However, treatment with Páramo soil yielded higher removal percentages. It was concluded that the axenic and heterotrophic cultures used with continuous flow feeding favored the increase in pH, the production of bioelectricity, and removal of organic matter. Keywords: Páramo Soils, Molecular Identification, Exo-Electrogenic Bacteria, Microbial Fuel Cell, Leachate Treatment DOI： https://doi.org/10.35741/issn.0258-2724.58.3.33

Heterotrophic culture of Spirulina platensis improved its growth and the study of its nutritional effect

With the development of human civilization, many people in the world still suffer from hunger due to wars, epidemics, and other causes such as not being able to afford a healthy diet. As a food, raw material with huge market potential, Spirulina platensis can be used as a new resource for food and has been in large-scale industrialized production worldwide. However, one of the biggest limitations to growing microalgae on an industrial scale is the high cost of the culturing medium. To reduce the culture cost, we hypothesized that S. platensis could grow better in a medium supplemented with glucose. In this study, we investigated the effects of Zarrouk’s medium, Central Food Technological Research Institute (CFTRI) medium, and CFTRI with glucose on the growth rate and biomass of S. platensis. Our results showed that compared with the expensive Zarrouk’s medium containing trace elements, adding a small amount of glucose to the low-cost CFTRI medium significantly increased S. platensis’s growth rate and biomass. Then, to further reduce breeding costs, we continued to explore the growth of S. platensis in a CFTRI medium supplemented with glucose under dark conditions. Our statistical data showed that the heterotrophic glucose consumption of S. platensis was affected by light, and the growth of S. platensis was limited in the absence of light. Finally, we used Drosophila melanogaster to verify the function of S. platensis and found that S. platensis supplementation increased longevity and reproductive ability in fruit flies.

Optimizing heterotrophic production of Chlorella sorokiniana SU-9 proteins potentially used as a sustainable protein substitute in aquafeed

Alternative protein sources for the reduction/replacement of fish meal in aqua-feeds are in urgent demand. Microalgae are considered sustainable protein sources for aquaculture due to their high-quality proteins with a complete profile of essential amino acids. This study examined the heterotrophic production of proteins from Chlorella sorokiniana SU-9. Culture parameters for maximal biomass and protein production are as follows: glucose – 10 g/L glucose, sodium nitrate – 1.5 g/L, and iron – 46 μM iron in BG-11 medium. Under optimal conditions, biomass content, protein content and protein productivity of SU-9 reached 4.14 ± 0.20 g/L, 403 ± 33 mg/g and 382 ± 36 mg/L/d, respectively. The protein profile of Chlorella sorokiniana SU-9 is comparable to fishmeal and soybean meal. The essential amino acids arginine, lysine and cysteine, along with glutamine and glutamate, were high. The production cost of SU-9 can be significantly reduced under heterotrophic cultivation conditions, making it a potential protein substitute in aquafeed.

Effects of directional adaptation on selenium tolerance and accumulation of heterotrophic Chlorella pyrenoidosa

Selenium (Se) is an essential trace element for organisms. Se deficiency will cause diseases such as Keshan disease and Kashin-Beck in human being, and huge loss to animal husbandry. Currently available Se supplements have such problems as low Se content, poor bioavailability, and poor safety. Chlorella pyrenoidosa can produce bioavailable and safe organic Se under suitable conditions, which is thus a promising Se supplement. Therefore, in this study, we tried to improve the Se tolerance and accumulation of C. pyrenoidosa by directional adaptation. To be specific, we gradually increased the concentration of Na2SeO3 in medium to domesticate C. pyrenoidosa and optimized the adapting time and concentration gradient of Na2SeO3 during the adaptation. The results showed that the adapted C. pyrenoidosa was more tolerant to Se and had stronger Se enrichment ability. In 5 L fermenter, the adapted strains could tolerate 40 mg/L Na2SeO3 and the synthesis rate of organic Se was 175.6% higher. Then, Se addition method in the 5 L fermenter was optimized. The result demonstrated that addition of Na2SeO3 at 40 mg/L during heterotrophic culture achieved the final dry weight of C. pyrenoidosa cells at 106.4 g/L, content of organic Se at 1 227 mg/kg, and synthesis rate of organic Se at 1.36 mg/(L·h). Compared with the reported highest cell density of 75 g/L and the highest organic Se content of 560 mg/kg, the corresponding figures in this study were 41.9% and 119.1% higher, respectively. In conclusion, directional adaptation can remarkably improve the Se tolerance and enrichment of C. pyrenoidosa.

Effects of shear stress and shear protectants on heterotrophic culture of Haematococcus pluvialis

A strategy named “Sequential Heterotrophy-Dilution-Photoinduction” is used for commercial production of astaxanthin in Haematococcus pluvialis. However, shear stress during heterotrophic culture has become the technical bottleneck for the wide application and amplification of this technology in microalgae culture, especially in the heterotrophic culture of H. pluvialis. To clarify the effects of shear stress, the influence of shear stress generated by different aeration rates on the heterotrophic growth of H. pluvialis was first investigated. The results showed that shear stress could cause the transformation of motile cells to non-motile cells, resulting in decrease in growth rate. The critical shear stress was produced by a superficial gas velocity of >3.21 × 10−3 m·s−1. The further analysis based on computational fluid dynamics indicated that shear stress-induced damage to cells was mainly caused by bubble rupture. To reduce the negative effects of shear stress, 0.05 % of Pluronic F-68 was selected for shear protection. Finally, it was successfully applied to the heterotrophic culture of H. pluvialis in a 500 L fermenter, increasing the cell yield by 30.39 %. This study could provide a reference and basis for the control of heterotrophic culture conditions and scale-up of H. pluvialis, and a feasible strategy for reducing the shear stress-induced damage during the heterotrophic process.

The growth and lipid accumulation of Scenedesmus quadricauda under nitrogen starvation stress during xylose mixotrophic/heterotrophic cultivation.

In order to conquer the block of high cost and low yields which limit to realize the commercialization of microalgal biodiesel, the mixotrophic and heterotrophic cultivation of Scenedesmus quadricauda FACHB-1297 fed on xylose was separately studied employing six forms of media: phosphorus sufficient, phosphorus restricted, and phosphorus starvation were combined with nitrogen sufficient and nitrogen starvation conditions. The maximum lipid content (about 41% of dry weight) was obtained on the 5th day (heterotrophic cultivation) and 8th day (mixotrophic cultivation) under the nitrogen starved and phosphorus sufficient (N0&P) conditions, which was about twofold in comparison to the final lipid content on the sufficient nitrogen condition (control). Under mixotrophic and heterotrophic modes, the highest lipid production was achieved in the N0&P trial, with the value of 274.96mg/L and 193.77mg/L, respectively. Xylose utilization rate of 30-96% under heterotrophic modes was apparently higher than that of 20-50% in mixotrophic modes. In contrast, phosphorus uptake rate of 100% under mixotrophic cultivation was significantly more than that of 60-90% in heterotrophic cultivation. Furthermore, under the condition of heterotrophic cultivation using xylose as a carbon source, the phosphorus had a positive impact on microalgae cell synthesis and the lipid content enhanced with the augmentation in phosphorus concentrations. We suggested that sufficient phosphorus should be supplied for obtaining higher microalgal lipid production in the lack of nitrogen under xylose heterotrophic/mixotrophic condition. This was a highly effective way to obtain efficient microalgae lipid production.

Simultaneous bioremediation of Disperse orange-2RL Azo dye and fatty acids production by Scenedesmus obliquus cultured under mixotrophic and heterotrophic conditions

Several types of green photosynthetic microalgae can grow through the process of heterotrophic growth in the dark with the help of a carbon source instead of the usual light energy. Heterotrophic growth overcomes important limitations in the production of valuable products from microalgae, such as the reliance on light, which complicates the process, raises costs, and lowers the yield of potentially useful products. The present study was conducted to explore the potential growth of green microalga Scenedesmus obliquus under mixotrophic and heterotrophic conditions utilizing Disperse orange 2RL Azo dye as a carbon source to produce a high lipid content and the maximum dye removal percentage. After 7 days of algal growth with dye under mixotrophic and heterotrophic conditions with varying pH levels (5, 7, 9, and 11), KNO3 concentrations (1, 1.5, 2, and 3 g/L), and dye concentrations (20, 40, and 60 ppm); dye removal percentage, algal dry weight, and lipid content were determined. The results showed that the highest decolorization of Disperse orange 2RL Azo dye (98.14%) was attained by S. obliquus in heterotrophic medium supplemented with glucose at the optimal pH 11 when the nitrogen concentration was 1 g/L and the dye concentration was 20 ppm. FT-IR spectroscopy of the dye revealed differences in peaks position and intensity before and after algal treatment. S. obliquus has a high concentration of oleic acid, which is enhanced when it is grown with Disperse orange 2RL Azo dye, making it ideal for production of high-quality biodiesel. In general, and in the vast majority of instances, heterotrophic cultivation is substantially less expensive, easier to set up, and requires less maintenance than mixotrophic cultivation. Heterotrophic cultivation allows for large-scale applications such as separate or mixed wastewater treatment along with biofuel production.

Thermal-tolerant potential of ordinary Chlorella pyrenoidosa and the promotion of cell harvesting by heterotrophic cultivation at high temperature.

During the heterotrophic cultivation of microalgae, a cooled process against temperature rise caused by the metabolism of exogenous organic carbon sources greatly increases cultivation cost. Furthermore, microalgae harvesting is also a cost-consuming process. Cell harvesting efficiency is closely related to the characteristics of the algal cells. It may be possible to change cell characteristics through controlling culture conditions to make harvesting easier. In this study, the mesophilic Chlorella pyrenoidosa was found to be a thermal-tolerant species in the heterotrophic mode. The cells could maintain their maximal specific growth rate at 40°C and reached 1.45day-1, which is equivalent to that of cultures at 35°C but significantly higher than those cultured at lower temperatures. Interestingly, the cells cultured at 40°C were much easier to be harvested than those at lower temperatures. The harvesting efficiency of the cells cultured at 40°C reached 96.83% after sedimentation for 240min, while the cells cultured at lower temperatures were reluctant to settle. Likely, the same circumstance occurred when cells were harvested by centrifugation or flocculation. The promotion of cell harvesting for cells cultured at high temperatures was mainly attributed to increased cell size and decreased cell surface charge. To the best of our knowledge, this is the first report that cells cultured at high temperatures can promote microalgae harvesting. This study explores a new approach to simplify the cultivation and harvesting of microalgae, which effectively reduces the microalgae production cost.

Co-production of lutein, zeaxanthin, and β-carotene by utilization of a mutant of the green alga Chromochloris zofingiensis

The unicellular green alga Chromochloris zofingiensis is a promising species for producing natural carotenoids. In this study, the mutant CZ-LZM3 was successfully obtained by screening the chemical mutagenesis pool of a C. zofingiensis strain. This mutant could achieve ultrahigh cell density and simultaneously accumulate lutein, zeaxanthin, and β-carotene under heterotrophic conditions. The insertional mutation was identified in the beta-carotene ketolase encoding gene, which may cause the block of astaxanthin biosynthesis in CZ-LZM3. Ultrahigh-cell-density of 185 g·L−1 was achieved under the optimum heterotrophic cultivation conditions in 7.5 L fermenters on Day 10, while the lutein, zeaxanthin, and β-carotene were 0.122, 0.22, 0.097 % of DW, respectively. By the combined utilization of gibberellin acid-3 (GA3) with the high C/N ratio and NaCl concentration under heterotrophic cultivation, the mutant could accumulate lutein, zeaxanthin, and β-carotene up to 0.726 % of DW. These findings demonstrated that the two-stage heterotrophic cultivation strategy is technically feasible for co-production of lutein, zeaxanthin, and β-carotene by utilizing the mutant of C. zofingiensis.

High-cell-density heterotrophic cultivation of microalga Chlorella sorokiniana FZU60 for achieving ultra-high lutein production efficiency

Chlorella sorokiniana has received particular attention as a promising candidate for microalgal biomass and lutein production. In this work, heterotrophic cultivation was explored to improve the lutein production efficiency of a lutein-rich microalga C. sorokiniana FZU60. Flask cultivation results showed that the highest lutein productivity was achieved at 30°C with an initial cell concentration of 1.40 g/L. Furthermore, six types of fed-batch strategies based on nutrient composition and concentration were examined using a 5 L fermenter. Among them, ultra-high lutein production (415.93 mg/L) and productivity (82.50 mg/L/d) with lutein content of 2.57 mg/g were achieved with fed-batch 3F (i.e., pulse-feeding with concentrated urea-N medium to achieve a 3-fold nutrient concentration). The lutein production performance achieved is much higher than the reported values. This work demonstrates that heterotrophic cultivation of C. sorokiniana FZU60 with the proposed fed-batch strategy could significantly enhance the production performance and the commercial viability of microalgae-derived lutein.

Production of biopolymer precursors beta-alanine and L-lactic acid from CO2 with metabolically versatile Rhodococcus opacus DSM 43205.

Hydrogen oxidizing autotrophic bacteria are promising hosts for conversion of CO2 into chemicals. In this work, we engineered the metabolically versatile lithoautotrophic bacterium R. opacus strain DSM 43205 for synthesis of polymer precursors. Aspartate decarboxylase (panD) or lactate dehydrogenase (ldh) were expressed for beta-alanine or L-lactic acid production, respectively. The heterotrophic cultivations on glucose produced 25 mg L−1 beta-alanine and 742 mg L−1 L-lactic acid, while autotrophic cultivations with CO2, H2, and O2 resulted in the production of 1.8 mg L−1 beta-alanine and 146 mg L−1 L-lactic acid. Beta-alanine was also produced at 345 μg L−1 from CO2 in electrobioreactors, where H2 and O2 were provided by water electrolysis. This work demonstrates that R. opacus DSM 43205 can be engineered to produce chemicals from CO2 and provides a base for its further metabolic engineering.

Heterotrophic Cultivation of Euglena gracilis in Stirred Tank Bioreactor: A Promising Bioprocess for Sustainable Paramylon Production.

Paramylon is a valuable intracellular product of the microalgae Euglena gracilis, and it can accumulate in Euglena cells according to the cultivation conditions. For the sustainable production of paramylon and appropriate cell growth, different bioreactor processes and industrial byproducts can be considered as substrates. In this study, a complex medium with corn steep solid (CSS) was used, and various bioreactor processes (batch, fed batch, semicontinuous and continuous) were performed in order to maximize paramylon production in the microalgae Euglena gracilis. Compared to the batch, fed batch and repeated batch bioprocesses, during the continuous bioprocess in a stirred tank bioreactor (STR) with a complex medium containing 20 g/L of glucose and 25 g/L of CSS, E. gracilis accumulated a competitive paramylon content (67.0%), and the highest paramylon productivity of 0.189 g/Lh was observed. This demonstrated that the application of a continuous bioprocess, with corn steep solid as an industrial byproduct, can be a successful strategy for efficient and economical paramylon production.

Zero-waste strategy by means of valorization of bread waste

To impose the zero-waste strategy through the diverse valorization route, this study used bread waste (BW) as the model compound. To realize these grand technical challenges, BW was enzymatically hydrolyzed to obtain a monomer sugar compound (glucose), and glucose was used as the carbon substrate in heterotrophic cultivation of Euglena gracilis ( E. gracilis ). Cultivation of E. gracilis using cultivation medium derived from BW was evaluated from a perspective of an economic viability . In addition, the more production of the target compound (paramylon, β-1,3-glucan) stemmed from E. gracilis was achieved (1.93 g L −1 d −1 ; 24% higher productivity than that of control). To approach zero waste disposal, bread waste residue (BWR) derived from enzymatic hydrolysis of BW was valorized into syngas . To offer a greener pyrolysis platform for BWR, CO 2 was used as a raw material. Here in this study, the mechanistic functionality of CO 2 was disclosed. In detail, CO 2 reacted with volatile matters (VMs) evolved from BWR, thereby resulting in CO 2 reduction. Simultaneously, reduced CO 2 also led to oxidation of VMs. Such consecutive gas-phase-reactions (GPRs) played a critical role to enhance CO formation. Lastly, the identified GPRs induced by CO 2 were tried to expedite the reaction kinetics in the presence of 5 wt% of Ni/SiO 2 catalyst. As a result, the molar concentrations of H 2 and CO in gaseous pyrolytic products derived from catalytic pyrolysis in CO 2 environment were 2- and 6-times higher, respectively, than that from pyrolysis without catalyst in N 2 environment. • Zero waste disposal platform for food waste (FW) was experimentally practiced. • Glucose derived from FW was used as carbon substrate for the growth of E. gracilis . • High yield of paramylon was achieved from E. gracilis grown with FW medium. • FW residue after glucose extraction was further valorized into energy. • CO 2 was converted into gaseous fuel in catalytic pyrolysis of FW residue.