Soy whey and brewer's spent grain hydrolysates wholly replace conventional medium for microalgae growth: Process performance and economic considerations.

To enhance circularity in heterotrophic microalgal bioprocesses, this study completely substituted glucose and Bold's basal medium (BBM) with brewer's spent grain (BSG) and soy whey (SW) hydrolysates. Mild acid hydrolysis conditions of BSG (0.2 M H2SO4, 130 °C, 36 min) and SW (0.1 M HCl, 95 °C, 30 min) were optimised for glucose release, and their hydrolysates were optimally mixed (15 % SW-85 % BSG) to obtain a medium that best supported Auxenochlorella protothecoides growth. Maximum biomass production (Xmax) and productivity (PXmax) obtained in the hydrolysate medium containing 50.75 g/L endogenous glucose (Xmax: 22.17 g/L; PXmax: 7.06 g/L/day) were comparable to that in BBM containing 50.44 g/L exogenous glucose (Xmax: 20.02 g/L; PXmax: 6.34 g/L/day). Moreover, estimated hydrolysate medium production costs were within an order of magnitude to BBM. Overall, the integrated approach of tailored hydrolytic treatments and complementary side-streams presents a promising technical and economic feasibility, with applications extending beyond A. protothecoides.

Enhancing protein extraction from heterotrophic Auxenochlorella protothecoides microalgae through emerging cell disruption technologies combined with incubation.

This study evaluated pulsed electric fields (PEF) and ultrasonication (US) combined with incubation to enhance cell disruption and protein extraction from Auxenochlorella protothecoides, comparing them to conventional high-pressure homogenization (HPH). A 5 h incubation enhanced protein yield by 79.4 % for PEF- and 27.2 % for US-treated samples. Extending the incubation to 24 h resulted in a total yield increase of 122 % for PEF (0.25 ± 0.03 kgEP kgTP-1) and 51.9 % for US (0.20 ± 0.02 kgEP-1 kgTP-1). Autofermentation in untreated cells after 24 h resulted in protein release with lower yields than all other treated and incubated samples. While HPH had the highest protein yield (0.58 ± 0.04 kgEP kgTP-1), PEF-incubation after 5 h (56.6 ± 5.3 MJ kgEP-1) and 24 h (49.5 ± 3.7 MJ kgEP-1) were 1.5 and 1.7-times more energy-efficient than HPH (82.9 ± 7.8 MJ kgEP-1). PEF combined incubation is an energy-efficient and targeted protein extraction method in heterotrophic A. protothecoides biorefinery.

Enhancing single-cell bioconversion efficiency by harnessing nanosecond pulsed electric field processing.

Nanosecond pulsed electric field (nsPEF) processing is gaining momentum as a physical means for single-cell bioconversion efficiency enhancement. The technology allows biomass yields per substrate (YX/S) to be leveraged and poses a viable option for stimulating intracellular compound production. NsPEF processing thus resonates with myriad domains spanning the pharmaceutical and medical sectors, as well as food and feed production. The exact working mechanisms underlying nsPEF-based enhancement of bioconversion efficiency, however, remain elusive, and a better understanding would be pivotal for leveraging process control to broaden the application of nsPEF and scale-up industrial implementation. To bridge this gap, the study provides the electrotechnological and metabolic fundamentals of nsPEF processing in the bio-based domain to enable a critical evaluation of pathways underlying the enhancement of single-cell bioconversion efficiency. Evidence suggests that treating cells during the rapid proliferating and thus the early to mid-exponential state of cellular growth is critical to promoting bioconversion efficiency. A combined effect of transient intracellular and sublethal stress induction and effects caused on the plasma membrane level result in an enhancement of cellular bioconversion efficiency. Congruency exists regarding the involvement of transient cytosolic Ca2+ hubs in nsPEF treatment responses, as well as that of reactive oxygen species formation culminating in the onset of cellular response pathways. A distinct assignment of single effects and their contributions to enhancing bioconversion efficiency, however, remains challenging. Current applications of nsPEF processing comprise microalgae, bacteria, and yeast biorefineries, but these endeavors are in their infancies with limitations associated with a lack of understanding of the underlying treatment mechanisms, an incomplete reporting, insufficient characterization, and control of processing parameters. The study aids in fostering the upsurge of nsPEF applications in the bio-based domain by providing a basis to gain a better understanding of cellular mechanisms underlying an nsPEF-based enhancement of cellular bioconversion efficiency and suggests best practice guidelines for nsPEF documentation for improved knowledge transfer. Better understanding and reporting of processes parameters and consequently improved process control could foster industrial-scale nsPEF realization and ultimately aid in perpetuating nsPEF applicability within the bio-based domain.

Automated Online Flow Cytometry Advances Microalgal Ecosystem Management as in situ, High-Temporal Resolution Monitoring Tool.

Microalgae are emerging as a next-generation biotechnological production system in the pharmaceutical, biofuel, and food domain. The economization of microalgal biorefineries remains a main target, where culture contamination and prokaryotic upsurge are main bottlenecks to impair culture stability, reproducibility, and consequently productivity. Automated online flow cytometry (FCM) is gaining momentum as bioprocess optimization tool, as it allows for spatial and temporal landscaping, real-time investigations of rapid microbial processes, and the assessment of intrinsic cell features. So far, automated online FCM has not been applied to microalgal ecosystems but poses a powerful technology for improving the feasibility of microalgal feedstock production through in situ, real-time, high-temporal resolution monitoring. The study lays the foundations for an application of automated online FCM implying far-reaching applications to impel and facilitate the implementation of innovations targeting at microalgal bioprocesses optimization. It shows that emissions collected on the FL1/FL3 fluorescent channels, harnessing nucleic acid staining and chlorophyll autofluorescence, enable a simultaneous assessment (quantitative and diversity-related) of prokaryotes and industrially relevant phototrophic Chlorella vulgaris in mixed ecosystems of different complexity over a broad concentration range (2.2-1,002.4 cells ⋅µL-1). Automated online FCM combined with data analysis relying on phenotypic fingerprinting poses a powerful tool for quantitative and diversity-related population dynamics monitoring. Quantitative data assessment showed that prokaryotic growth phases in engineered and natural ecosystems were characterized by different growth speeds and distinct peaks. Diversity-related population monitoring based on phenotypic fingerprinting indicated that prokaryotic upsurge in mixed cultures was governed by the dominance of single prokaryotic species. Automated online FCM is a powerful tool for microalgal bioprocess optimization owing to its adaptability to myriad phenotypic assays and its compatibility with various cultivation systems. This allows advancing bioprocesses associated with both microalgal biomass and compound production. Hence, automated online FCM poses a viable tool with applications across multiple domains within the biobased sector relying on single cell-based value chains.

Nanosecond pulsed electric field processing of microalgae based biorefineries governs growth promotion or selective inactivation based on underlying microbial ecosystems.

Nanosecond pulsed electric field treatment (nsPEF) is a technology-driven, resource-efficient approach fostering microalgae biorefineries for transforming them into economically viable scenarios. A processing window of 100 ns, 7 Hz, and 10 kV cm-1 significantly leveraged phototrophic Chlorella vulgaris and bacterial counts up to + 50.1 ± 12.2% and + 77.0 ± 37.4%, respectively (n = 4; p < 0.05) in non-axenic cultures. Applying the same processing window decreased C. vulgaris (-17.1 ± 13.8%) and prokaryotic (-82.7 ± 14.6%) counts owing to alterations in the prokaryotic community diversity. Principle coordinate analysis of prokaryotic phenotypic fingerprints indicated that phenotype or metabolism related diversity changes in the prokaryotic community affected the treatment outcome. The study fosters the upsurge of industrial-scale nsPEF realization and the economic viability of microalgae biorefineries through improved process understanding and thus control. It perpetuates nsPEF applicability for microalgae feedstock production and several other applications within single-cell biorefineries in the bio-based domain.

Characterization of Chlorella vulgaris (Trebouxiophyceae) associated microbial communities1.

Microalgae exhibit extensive potential for counteracting imminent challenges in the nutraceutical, pharmaceutical, and biomaterial sectors, but lack economic viability. Biotechnological systems for contamination control could advance the economic viability of microalgal feedstock, but the selection of suitable strains that sustainably promote microalgal productivity remains challenging. In this study, total diversity in phototrophic Chlorella vulgaris cultures was assessed by amplicon sequencing comparing cultures subjected to five different cultivation conditions. Overall, 12 eukaryotic and 53 prokaryotic taxa were identified; Alphaproteobacteria (36.7%) dominated the prokaryotic and C. vulgaris (97.2%) the eukaryotic community. Despite altering cultivation conditions, 2 eukaryotic and 40 prokaryotic taxa remained stably associated with C. vulgaris; diversity between systems did not significantly differ (P > 0.05). Among those, 20 cultivable taxa were isolated and identified by 16S rDNA sequencing. Subsequently, controlled co-cultures were investigated showing stable associations of C. vulgaris with Sphingopyxis sp. and Pseudomonas sp.. Out-competition of C. vulgaris due to ammonium or phosphate limitation was not observed, despite significantly elevated growth of Sphingopyxis sp. and Tistrella sp.. (P < 0.05). Nevertheless, C. vulgaris growth was impaired by Tistrella sp.. Hence, the study provides a selection of stable indigenous prokaryotes and eukaryotes for artificially tailoring microbial biocenoses. Following a bottom-up approach, it provides a base for controlled co-cultures and thus the establishment of even more complex biocenoses using interkingdom assemblages. Such assemblages can benefit from functional richness for improved nutrient utilization, as well as bacterial load control, which can enhance microalgal feedstock production through improved culture stability and productivity.

Continuous nanosecond pulsed electric field treatments foster the upstream performance of Chlorella vulgaris-based biorefinery concepts.

Nanosecond pulsed electric field treatment (nsPEF) is an innovative, technology-driven, and resource-efficient approach to foster the upstream performance of microalgae-based biorefinery concepts to transform microalgae into economic more viable raw materials for the biobased industry. A processing window applying three treatments of 100 ns, 5 Hz, and 10 kV cm-1 to industrially relevant phototrophic Chlorella vulgaris in the early exponential growth phase significantly increased biomass yields by up to 17.53 ±â€¯10.46% (p = 3.18 × 10-5). Treatments had limited effects on the carbon and pigment contents, but the protein content was decreased. The longest possible pulse width (100 ns) resulted in the highest biomass yield indicating underlying working mechanisms of enhanced cell proliferation based on intracellular and plasma membrane-related effects. The applicability to eukaryotes and prokaryotes, such as C. vulgaris and cyanobacteria highlights the possible impacts of nsPEF across multiple domains of the biobased industry relying on single-cell-based value-chains.

Pulsed electric field based cyclic protein extraction of microalgae towards closed-loop biorefinery concepts.

Microalgae-based biorefinery concepts can contribute to providing sufficient resources for a growing world population. However, the performance needs to be improved, which requires innovative technologies and processes. Continuous extraction from Chlorella vulgaris cultures via pulsed electric field (PEF) processing might be a viable process to increase the performance of microalgae-based biorefinery concepts. In this study, increasing protein extraction rates were observed with increasing electric field strength, up to 96.6 ±â€¯4.8% of the free protein in the microalgae. However, increased extraction rates negatively influenced microalgae growth after PEF treatment. A free protein extraction rate up to 29.1 ±â€¯1.1% without a significant influence on microalgal growth after 168 h was achieved (p = 0.788). Within the scope of this work, a protocol for continuous protein extraction during microalgae cultivation by PEF processing was developed. The incorporation of innovative downstream into upstream processing could be a viable future concept.

Cultivation of Chlorella protothecoides under different growth modes and its utilisation in oil/water emulsions.

Microalgae can be incorporated in different bio-based products; however, the green colour is a barrier for a successful integration. This study aims to overcome this barrier by growing microalgae in different cultivation modes. Mixotrophic cultivation of Chlorella protothecoides resulted in the highest biomass production after 5 days (5.56 ±â€¯0.09 g/L), followed by heterotrophic and photoautotrophic cultivation (4.33 ±â€¯0.15 and 1.80 ±â€¯0.05 g/L, respectively). Mixotrophically and heterotrophically produced biomass presented a reduced greenish colouration compared to photoautotrophically produced biomass. Chlorophyll content resulted in 1.46 ±â€¯0.21 and 0.95 ±â€¯0.28 mg/g dry weight (DW) in mixotrophic and heterotrophic cultures, respectively, and 25.98 ±â€¯1.28 mg/g DW in photoautotrophic cultures. In contrast, the fraction of carotenoids in the total pigments was much higher. With the whole microalgae fractions after cell disruption as ingredients, stable emulsions containing 50% oil could be produced. No syneresis with serum separation was observed 24 h after preparation.