Access the full text.
Sign up today, get DeepDyve free for 14 days.
Eric Poliner, Jane Pulman, K. Zienkiewicz, K. Childs, C. Benning, E. Farré (2017)
A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid productionPlant Biotechnology Journal, 16
Isabel Espinosa-Gonzalez, A. Parashar, D. Bressler (2014)
Heterotrophic growth and lipid accumulation of Chlorella protothecoides in whey permeate, a dairy by-product stream, for biofuel production.Bioresource technology, 155
J Bentahar (2022)
1479Can J Chem Eng, 100
M. Zwietering, I. Jongenburger, F. Rombouts, K. Riet (1990)
Modeling of the Bacterial Growth CurveApplied and Environmental Microbiology, 56
D. Correia, L. Dias, A. Veloso, T. Dias, I. Rocha, L. Rodrigues, A. Peres (2014)
Dietary Sugars Analysis: Quantification of Fructooligossacharides during Fermentation by HPLC-RI MethodFrontiers in Nutrition, 1
Zhang Tian-yuan, Wu Yin-hu, Zhuang Lin-Lan, Wang Xiao-Xiong, H. Hong-ying (2014)
Screening heterotrophic microalgal strains by using the Biolog method for biofuel production from organic wastewaterAlgal Research-Biomass Biofuels and Bioproducts, 6
E. Daneshvar, Mohammad Zarrinmehr, Atefeh Hashtjin, O. Farhadian, A. Bhatnagar (2018)
Versatile applications of freshwater and marine water microalgae in dairy wastewater treatment, lipid extraction and tetracycline biosorption.Bioresource technology, 268
Rita Branco, M. Amândio, L. Serafim, A. Xavier (2020)
Ethanol Production from Hydrolyzed Kraft Pulp by Mono- and Co-Cultures of Yeasts: The Challenge of C6 and C5 Sugars ConsumptionEnergies
J. Knowles (1980)
Enzyme-catalyzed phosphoryl transfer reactions.Annual review of biochemistry, 49
Shengping You, Xiao-nan Wang, W. Qi, R. Su, Zhimin He (2017)
Optimisation of culture conditions and development of a novel fed‐batch strategy for high production of &bgr;‐galactosidase by Kluyveromyces lactisInternational Journal of Food Science & Technology, 52
R. Halim, D. Hill, E. Hanssen, P. Webley, Susan Blackburn, A. Grossman, C. Posten, Gregory Martin (2019)
Towards sustainable microalgal biomass processing: anaerobic induction of autolytic cell-wall self-ingestion in lipid-rich Nannochloropsis slurriesGreen Chemistry
Wei-hsin Chen, B. Lin, Ming-Yueh Huang, Jo‐Shu Chang (2015)
Thermochemical conversion of microalgal biomass into biofuels: a review.Bioresource technology, 184
H. Kiani, Yeganeh Azimi, Yuchen Li, M. Mousavi, Fanny Cara, S. Mulcahy, Hugh McDonnell, A. Blanco, R. Halim (2022)
Nitrogen and phosphate removal from dairy processing side-streams by monocultures or consortium of microalgae.Journal of biotechnology
Lydia Mapstone, H. Taunt, Jing Cui, S. Purton, Tom Brooks (2022)
ADA: an open-source software platform for plotting and analysis of data from laboratory photobioreactorsApplied Phycology, 3
D. Otieno (2010)
Synthesis of β-Galactooligosaccharides from Lactose Using Microbial β-Galactosidases.Comprehensive reviews in food science and food safety, 9 5
H. Lawford, J. Rousseau (1996)
Studies on nutrient requirements and cost-effective supplements for ethanol production by recombinantE. coliApplied Biochemistry and Biotechnology, 57-58
Songhe Zhang, S. Pang, Peifang Wang, Chao Wang, Chuan Guo, Felix Addo, Yi Li (2016)
Responses of bacterial community structure and denitrifying bacteria in biofilm to submerged macrophytes and nitrateScientific Reports, 6
A. Wood, D. Kelly, F. Rainey (2002)
The genus paracoccus
M. Carević, M. Vukasinovic-Sekulic, Sanja Grbavčić, M. Stojanovic, Mladen Mihailović, A. Dimitrijević, Dejan Bezbradica (2014)
Optimization of β-galactosidase production from lactic acid bacteriaHemijska Industrija, 69
Jéssika Zimermann, E. Sydney, M. Cerri, Isabella Carvalho, Kathlyn Schafranski, A. Sydney, Luciano Vitali, S. Gonçalves, G. Micke, C. Soccol, I. Demiate (2020)
Growth kinetics, phenolic compounds profile and pigments analysis of Galdieria sulphuraria cultivated in whey permeate in shake-flasks and stirred-tank bioreactorJournal of water process engineering, 38
L. Robertson, J. Kuenen (2004)
Aerobic denitrification: a controversy revivedArchives of Microbiology, 139
H. Togt (2003)
Publisher's NoteJ. Netw. Comput. Appl., 26
M. Hemalatha, J. Sravan, B. Min, S. Mohan (2019)
Microalgae-biorefinery with cascading resource recovery design associated to dairy wastewater treatment.Bioresource technology, 284
S. Chniti, M. Jemni, Imène Bentaha, M. Shariati, Y. Kadmi, Hayet Djelal, A. Amrane, M. Hassouna, H. Elmsellem (2017)
Kinetic of sugar consumption and ethanol production on very high gravity fermentation from syrup of dates by- products (Phoenix dactylifera L.) by using Saccharomyces cerevisiae, Candida pelliculosa and Zygosaccharomyces rouxiiThe Journal of Microbiology, Biotechnology and Food Sciences, 7
Chris Hulatt, R. Wijffels, S. Bolla, V. Kiron (2017)
Production of Fatty Acids and Protein by Nannochloropsis in Flat-Plate PhotobioreactorsPLoS ONE, 12
A. Slavov (2017)
General Characteristics and Treatment Possibilities of Dairy Wastewater - A Review.Food technology and biotechnology, 55 1
Richard Smith, K. Bangert, Stephen Wilkinson, D. Gilmour (2015)
Synergistic carbon metabolism in a fast growing mixotrophic freshwater microalgal species Micractinium inermumBiomass & Bioenergy, 82
A. Montilla (2020)
Review for "Influence of sweet whey permeate utilization on Tetradesmus obliquus growth and β‐galactosidase production"
Jihed Bentahar, A. Doyen, L. Beaulieu, J. Deschênes (2018)
Investigation of β-galactosidase production by microalga Tetradesmus obliquus in determined growth conditionsJournal of Applied Phycology, 31
C. Zanette, A. Mariano, Yuri Yukawa, Israel Mendes, Michele Spier (2019)
Microalgae mixotrophic cultivation for β-galactosidase productionJournal of Applied Phycology, 31
Zhao Zhang, Dongzhe Sun, Ka-Wing Cheng, Feng Chen (2021)
Investigation of carbon and energy metabolic mechanism of mixotrophy in Chromochloris zofingiensisBiotechnology for Biofuels, 14
L. Hazleton (1967)
Food Chemicals CodexJournal of Pharmaceutical Sciences, 56
E. Daneshvar, Mohammad Zarrinmehr, E. Koutra, M. Kornaros, O. Farhadian, A. Bhatnagar (2019)
Sequential cultivation of microalgae in raw and recycled dairy wastewater: Microalgal growth, wastewater treatment and biochemical composition.Bioresource technology, 273
R. Halim, D. Hill, E. Hanssen, P. Webley, G. Martin (2019)
Thermally coupled dark-anoxia incubation: A platform technology to induce auto-fermentation and thus cell-wall thinning in both nitrogen-replete and nitrogen-deplete Nannochloropsis slurries.Bioresource technology, 290
Nature Poddar, R. Sen, G. Martin (2018)
Glycerol and nitrate utilisation by marine microalgae Nannochloropsis salina and Chlorella sp. and associated bacteria during mixotrophic and heterotrophic growthAlgal Research
Jihed Bentahar, A. Doyen, L. Beaulieu, J. Deschênes (2019)
Acid whey permeate: An alternative growth medium for microalgae Tetradesmus obliquus and production of β-galactosidaseAlgal Research
W. Kolanowski (2021)
Salmonids as Natural Functional Food Rich in Omega-3 PUFAApplied Sciences
Gentoku Nakase, M. Eguchi (2007)
Analysis of bacterial communities in Nannochloropsis sp. cultures used for larval fish productionFisheries Science, 73
Shyam Suwal, Jihed Bentahar, A. Marciniak, L. Beaulieu, J. Deschênes, A. Doyen (2019)
Evidence of the production of galactooligosaccharide from whey permeate by the microalgae Tetradesmus obliquusAlgal Research
Fengwei Yin, Si-yu Zhu, Dong‐Sheng Guo, Lu-jing Ren, Xiaojun Ji, He Huang, Zhen Gao (2019)
Development of a strategy for the production of docosahexaenoic acid by Schizochytrium sp. from cane molasses and algae-residue.Bioresource technology, 271
Yong-Keun Choi, H. Jang, E. Kan (2018)
Microalgal Biomass and Lipid Production on Dairy Effluent Using a Novel Microalga, Chlorella sp. Isolated from Dairy WastewaterBiotechnology and Bioprocess Engineering, 23
R. Guillard, J. Ryther (1962)
Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran.Canadian journal of microbiology, 8
E. Jurado, F. Camacho, G. Luzón, J. Vicaria (2004)
Kinetic models of activity for β-galactosidases: influence of pH, ionic concentration and temperatureEnzyme and Microbial Technology, 34
J. Girard, M. Roy, M. Hafsa, J. Gagnon, N. Faucheux, M. Heitz, R. Tremblay, J. Deschênes (2014)
Mixotrophic cultivation of green microalgae Scenedesmus obliquus on cheese whey permeate for biodiesel productionAlgal Research-Biomass Biofuels and Bioproducts, 5
Thi Doan, J. Obbard (2011)
Improved Nile Red staining of Nannochloropsis sp.Journal of Applied Phycology, 23
T. Silva, Patrícia Moniz, Carla Silva, A. Reis (2021)
The Role of Heterotrophic Microalgae in Waste Conversion to Biofuels and BioproductsProcesses
Tethi Biswas, S. Bhushan, Sanjeev Prajapati, S. Chaudhuri (2021)
An eco-friendly strategy for dairy wastewater remediation with high lipid microalgae-bacterial biomass production.Journal of environmental management, 286
G. Gramegna, A. Scortica, V. Scafati, F. Ferella, L. Gurrieri, M. Giovannoni, R. Bassi, F. Sparla, B. Mattei, M. Benedetti (2020)
Exploring the potential of microalgae in the recycling of dairy wastesBioresource Technology Reports
D. Sahin, Ulku Altindag, Ezgi Taş (2018)
Enhancement of docosahexaenoic acid (DHA) and beta-carotene production in Schizochytrium sp. using symbiotic relationship with Rhodotorula glutinisProcess Biochemistry
K. Handley, J. Lloyd (2013)
Biogeochemical implications of the ubiquitous colonization of marine habitats and redox gradients by Marinobacter speciesFrontiers in Microbiology, 4
Tomasz Półbrat, Damian Konkol, M. Korczyński (2021)
Optimization of docosahexaenoic acid production by Schizochytrium SP. – A reviewBiocatalysis and Agricultural Biotechnology
Ying Liu, Guo-Min Ai, L. Miao, Zhipei Liu (2016)
Marinobacter strain NNA5, a newly isolated and highly efficient aerobic denitrifier with zero N2O emission.Bioresource technology, 206
This study investigated the mechanism of lactose assimilation in Nannochloropsis oceanica for dairy-wastewater bioreme- diation and co-production of valuable feed/food ingredients in a circular dairy system (β-galactosidase and omega-3 poly- unsaturated fatty acids). Mixotrophic cultivation was found to be mandatory for lactose assimilation in N. oceanica, with biomass production in mixotrophic cultures reaching a fourfold increase over that under heterotrophic conditions. Under mixotrophic conditions, the microalgae were able to produce β-galactosidase enzyme to hydrolyse lactose, with maximum −1 extracellular secretion recorded on day 8 of growth cycle at 41.47 ± 0.33 U g . No increase in the concentration biomass of glucose or galactose was observed in the medium, confirming the ability of microalgae to indiscriminately absorb the resultant monosaccharides derived from lactose breakdown. Population analysis revealed that microalgae cells were able to maintain dominance in the mixotrophic culture, with bacteria accounting for < 12% of biomass. On the other hand, under heterotrophic conditions, native bacteria took over the culture (occupying over 95% of total biomass). The bacteria, how- ever, were also unable to effectively assimilate lactose, resulting in limited biomass increase and negligible production of extracellular β-galactosidase. Results from the study indicate that N. oceanica can be effectively applied for onsite dairy wastewater treatment under strict mixotrophic conditions. This is commercially disadvantageous as it rules out the possibility of deploying heterotrophic fermentation with low-cost bioreactors and smaller areal footprint. Keywords Lactose · β-galactosidase · Microalgae · Mixotrophy · Dairy, Nannochloropsis Introduction Dairy processing and cheese production generate significant amount of wastewater and side products which are rich in lactose (up to 80 wt% of dissolved solids), other carbon- based nutrients (e.g., proteins and lipids) and inorganic * Ronald Halim nutrients (e.g., nitrates, phosphates and micronutrients) ronald.halim@ucd.ie (Slavov 2017; Daneshvar et al. 2019). These waste products 1 pose signic fi ant environmental problems due to high organic School of Biosystems and Food Engineering, University matter contents, BOD level and COD level (Gramegna et al. College Dublin, Belfield, Dublin 4, Ireland 2 2020), and are generally subjected to conventional waste- Conway Institute, University College Dublin, water treatment requiring large amounts of chemicals and Belfield, Dublin 4, Ireland 3 energy prior to disposal. Wastewater treatment represents Department of Food Chemistry and Technology, Ashtown a significant economic burden to dairy manufacturers and Teagasc Food Research Centre, Dublin 15, Ireland 4 successful valorisation of dairy waste streams into viable School of Chemistry, University College Dublin, and profitable new co-products is of critical importance Belfield, Dublin 4, Ireland 5 (Espinosa-Gonzalez et al. 2014). In the present, however, Arrabawn Co-Operative Society Ltd., Nenagh, Co. Tipperary, attempts to commercially use dairy side streams (e.g., whey Ireland Vol.:(0123456789) 1 3 Journal of Applied Phycology permeate) have not been highly profitable, with the perme- to monosaccharide sub-units prior to metabolization, result- ate either sold directly as low-value livestock feed without ing in a more intricate assimilation process compared to significant profit or converted into products (e.g., lactose monosaccharides (such as glucose). Yet studies evaluating powder or alcoholic beverages) with relatively low market the use of microalgae for dairy-waste bioremediation have values. overlooked this aspect, directly assessing the performance Microalgae can serve as an attractive valorisation strategy of microalgae in removing nutrients from dairy wastewater for dairy by-products. Dairy side streams, such as whey perme- without having any understanding of how lactose availabil- ate, can be used as a low-cost carbon and nutrient-rich medium ity and breakdown can affect the bioremediation kinetics of in mixotrophic and heterotrophic cultivation of microalgae other nutrients (such as phosphates and nitrates). This has (Zimermann et al. 2020; Bentahar and Deschênes 2022). The resulted in inconsistent performance reported for microalgae bioremediation of dairy streams by algal cells will neutralise treatment of dairy waste, with wide-ranging nutrient removal nutrients in a sustainable manner (compared to chemical- efficiency from 30 to 100% (Daneshvar et al. 2018; Biswas intensive wastewater treatment) while simultaneously gener- et al. 2021; Kiani et al. 2023). ate biomass which can be commercially exploited for food and Amongst the few studies to-date that have investigated bioactive compounds (Espinosa-Gonzalez et al. 2014; Benta- microalgae utilization of lactose (Suwal et al. 2019; Zanette har et al. 2019a). Species belonging to the Nannochloropsis et al. 2019; Bentahar and Deschênes 2022), to the best of our genus are particularly attractive for dairy-effluent valorisation knowledge, only one study has investigated the mechanism because of their high content of omega-3 polyunsaturated fatty of lactose assimilation in Nannochloropsis. The capacity of acids (ω-3 PUFAs) in the form of eicosapentaenoic acid or EPA Nannochloropsis to assimilate lactose under heterotrophic (up to ca. 10 wt% of biomass) (Hulatt et al. 2017; Halim et al. regimes has never been explored. Heterotrophic fermenta- 2019a). ω-3 PUFAs are ubiquitously used in the food and feed tion has several key advantages compared to an autotrophic industry (including dairy industry) as nutrient enrichment to system in the context of dairy waste bioremediation; it elimi- promote immunity and brain development (e.g., in infant for- nates dependence on light irradiation which can often be mulas) (Kolanowski 2021). A sustainable alternative source of limiting in northern Europe and reduces areal requirements ω-3 PUFAs is, however, urgently needed as commercial pro- through simplification of bioreactor design. Standard pho - duction currently relies on fish oil derived from wild-caught fish tobioreactors used for autotrophic/mixotrophic cultivation and places enormous strain on the oceanic fish stock (Poliner are constructed with large surface area to volume ratio to et al. 2018). While alternative docosahexaenoic acid (DHA) enable light penetration, naturally requiring more land foot- from Schizochytrium is already commercially available (Sahin print compared to heterotrophic fermenters (Tian-Yuan et al. et al. 2018; Yin et al. 2019; Półbrat et al. 2021), there is still no 2014; da Silva et al. 2021). Commercial dairy manufactur- commercial source for EPA not derived from fishing. Nanno- ers generally have limited space to expand into and cannot chloropsis can therefore exert dual environmental impact for the afford to implement on-site wastewater solutions requiring dairy processing industry, providing a more sustainable means large areal footprint. for wastewater treatment while producing alternative EPA that In addition, the interaction between native bacteria can then be recycled within the industry in a circular manner (defined as symbiotic/associated bacteria present in the to reduce demand for conventional fish oil. algal culture since inoculation) and Nannochloropsis The application of Nannochloropsis in treating dairy cells during mixotrophic and heterotrophic cultivation waste can also lead to the production of β-galactosidase (or and their effect on carbon assimilation has not been lactase), a key enzyme for the synthesis of galactooligo- elucidated, bringing uncertainty to the behaviour of saccharides (GOS) prebiotic compounds in infant formulas culture under non-axenic conditions encountered in (Suwal et al. 2019), thus adding an extra layer of commercial large-scale wastewater treatment. Finally, the dependence benefits to the dairy industry. Despite these potential ben- of β-galactosidase production in Nannochloropsis on efits, only few studies have explored the use of Nannochlo- growth conditions and stages of cultivation is not well ropsis for the bioremediation of dairy waste (Zanette et al. understood. 2019; Kiani et al. 2023). This study aims to shed light on the mechanism of lac- Fundamental understanding of the mechanism respon- tose assimilation in N. oceanica to harness its potential for sible for lactose assimilation in microalgae is critical to dairy-effluent bioremediation and co-generation of valu- the design and optimisation of microalgae systems for able EPA and β-galactosidase. The study plans to derive dairy-waste valorisation and the transfer of results from three key insights regarding N. oceanica’s lactose assimi- bench-scale to industrial scale. Lactose is a disaccharide lation: 1) the capacity of the species to assimilate lactose composed of glucose and galactose sub-units. It is structur- under both mixotrophic and heterotrophic conditions, 2) ally complex and will likely require the microalgae cells to the interaction of N.oceanica cells with native bacteria and produce β-galactosidase enzyme to hydrolyse the molecule its effect on lactose assimilation under different cultivation 1 3 Journal of Applied Phycology regimes and 3) the extent of β-galactosidase production and Biomass production and growth parameters the dependence of its kinetics on lactose concentration. To achieve these objectives, the study cultivated N. oceanica Optical density (OD) of each culture was measured at on lactose-rich standard solution of different initial lactose 750 nm using UV-Vis spectrophotometer (Spectramax M3, concentrations under both mixotrophic and heterotrophic Molecular devices, Berkshire, UK) every two days. The conditions and compared their biomass production and lac- OD values were then converted to biomass concentra- tose assimilation. The levels of extracellular β-galactosidase tion to monitor the growth of microalgae using a previously secretion as well as lactose breakdown products (i.e., glu-determined OD -biomass concentration calibration curve cose and galactose) in the medium of different cultures were for the strain. OD measurement for each collected sample monitored to study the kinetics of β-galactosidase evolution was carried out in duplicate. −1 and determine the mechanism of lactose assimilation. Flow The specific growth rate (µ, day ) was calculated using an −1 cytometry was employed throughout the study to investigate equation between biomass concentration (Y, g L ) and cultiva- the cell population dynamics in the medium and quantita- tion time (t, day) based on the model of Zwietering et al. (1990). tively measure the level of bacteria in the cultures, allow- ing for the determination of their roles in β-galactosidase Y = (1) synthesis and lactose assimilation. Finally, carbon mass bal- 1 + exp ( − t) + 2 ance around the different cultures was conducted to under - −1 stand the effect of cultivation modes on microalgal carbon where Y is the maximum biomass concentration (g L ), µ m m −1 metabolism. is the maximum specific growth rate (day ) and the λ is lag time (day). All curve-fitting calculations were performed using Algal Data Analyser (ADA) software (Mapstone et al. 2022). Materials and methods Chemical analysis Microalgae strain and culture conditions Ten mL of supernatant was collected from each culture every Microalgae strain was obtained from the Culture Collec- two days by centrifugation at 4000 rpm for 10 min, which was tion of Algae and Protozoa (CCAP, Scottish Association used for the following analyses. Colorimetric methods were for Marine Science, Oban, Scotland, U.K.). Nannochlo- employed to quantify the nitrate and phosphate concentrations ropsis oceanica (CCAP849/10 isolated from operational in the supernatant using commercial kits according to suppli- hatchery western Norway; non-axenic stock culture) was ers’ instructions. The analysis kits for nitrate and phosphate assessed in this study. Mixotrophic and heterotrophic cul- were obtained from API (Mars Fishcare, USA) and Red Sea tivation of N. oceanica was performed with f/2 medium (Seahorse, Ireland), respectively. Chemical analyses for each (Guillard and Ryther 1962) in synthetic seawater (Red collected sample were carried out in duplicate. Sea Coral Pro Salt, Rea Sea, USA). Lactose solutions Nitrate and phosphate reduction (%) was calculated using (α-Lactose monohydrate, CAS No. 5989–81-1, Merck Eq. 2 and 3. KGaA, Germany) were separately prepared, autoclaved, C − C N0 N and added to the f/2 medium previously prepared under Nitrate reduction (%) = × 100 (2) −1 sterile conditions to form culture media with 10 g L N0 −1 (or 1% (w/v)) and 5 g L (or 0.5% (w/v)) initial lactose concentration. Cultures without sugar addition were used C − C P0 P Phosphate reduction (%) = × 100 (3) as a control group (i.e., autotrophic control and dark con- P0 trol), leading to a total of six independent experiments where C and C are the nitrate and phosphate concentra- (Table 1). The autotrophic and mixotrophic samples were N P tions (ppm) at time t, and C and C are the initial concen- cultivated in 250 mL Erlenmeyer flasks with a 150 mL N0 P0 tration of the nitrate and phosphate in the culture. working volume under constant orbital shaking (130 rpm) on a 12 h:12 h light:dark cycle at ambient temperature (22 °C ± 1 °C) without additional aeration for 30 days. HPLC analysis Cotton caps were placed on each flask to reduce con- tamination and evaporation of the growth medium during The concentrations of lactose, glucose and galactose in the cultivation period. The dark-control and heterotrophic the supernatants were determined by high-performance samples were cultivated under the same conditions with liquid chromatography (HPLC). The chromatographic no illumination. All cultivations were performed in separation was achieved using an Agilent ZORBAX NH duplicate. (250 mm × 4.6 mm containing 5 μm silica particles) column 1 3 Journal of Applied Phycology in an Agilent Technologies 1200 Series equipped with a after staining by excitation at 450 nm for DAPI. In addi- refractive index detector (G1362A RID) at 35 °C. A mix- tion, forward scatter (FSC) and side scatter (SSC) were ture of acetonitrile and 0.04% ammonium hydroxide in water measured as indicators of the diameter (or size) and the (70:30, v/v) at pH 6.92 ± 0.05 was employed for isocratic density of the cell, respectively. The data were subse- −1 elution at a flow rate of 1.5 mL min (Correia et al. 2014). quently analysed with CytoFLEX software to determine The running time of each sample was 11 min with a 10 µL the algal cell population dynamics. Algal and bacteria injection volume and a column temperature at 30 °C. The biomass concentrations were calculated as in Eq. 4 and sugars were identified by retention time and quantified by 5 respectively. comparing their peak areas to a linear calibration curve of Y × FSC × SSC × N known standards. Y = Microalgae (4) FSC × SSC ×N A A A β‑Galactosidase activity assay Y = Y − Y Bacteria Microalgae (5) The β-galactosidase activity was measured using where Y and Y are the contribution to biomass microalgae Bacteria o-nitrophenyl-β-D-galactopyranoside (ONPG) as a sub- concentrations in the culture by algal cell and by bacteria strate based on the method reported by National Research −1 −1 cells, respectively (g L ), N is algal cell count (cells mL ) Council (1981), with some modifications as described by detected by flow cytometer, FSC and SSC are forward scatter Suwal et al. (2019) and Zanette et al. (2019). ONPG solu- median and side scatter median of algal cells, respectively. −1 tion (4 mg mL ) was prepared in 0.1 M sodium phos- The subscript A represents results of autotrophic cultures. phate buffer (same pH as the supernatant). Then 1 mL of This calculation is based on a hypothesis that only algal cells supernatant was added into 2 mL of ONPG solution and contributed to biomass concentration in autotrophic cultures. incubated at 30 °C for 15 min. The reaction was stopped by adding 1 mL of 1 M N a CO . The absorbance against 2 3 distilled water at 420 nm was recorded. The enzyme activ- Carbon flow −1 ity, expressed as specific activity (U g ), was calcu- biomass lated based on a standard curve. One unit of β-galactosidase Carbon mass balance around each cultivation system was activity (U) was defined as the amount of enzyme that liber - determined in this study. The quantity of carbon stored ated 1 μmol of o-nitrophenol per minute. The measurement in both microalgae and bacteria biomass (M, g C-biomass C −1 of β-Galactosidase activity for each collected sample was L ) and carbon assimilated from lactose (M g C-lactose, C −1 carried out in duplicate. L ) were calculated as in Eq. 6 and 7. M = × Y (6) C−biomass B Flow cytometry M = × C − C (7) C−lactose L L0 L The culture was sampled every seven days for flow cytometry analysis. The sample (2 mL) was first har- where η (η = 50.06%) and η (η = 42.11%) are the per- B B L L vested by centrifugation at 4000 rpm for 10 min and centage of carbon content of algal-bacteria consortium bio- the supernatant carefully removed. Then 4 mL Phos- mass (Lawford and Rousseau 1996; Chen et al. 2015) and phate Buffered Saline (PBS, pH 7.4) was added to the lactose respectively, C is the lactose concentration, C is L L0 cells and mixed thoroughly using a vortex mixer. DAPI the initial lactose concentration of the culture. (4′,6-diamidino-2-phenylindole) was used to stain the Then the carbon mass balance was calculated using Eq. 8. nucleus of both microalgae and bacteria cells in the M =M +M microalgal suspension. The dye was first dissolved in C−biomass C−lactose C−capture (8) −1 anhydrous dimethyl sulfoxide (DMSO) at 10 μL mL to where M is the mass of carbon captured during cul- C-capture enhance the staining efficacy for nucleus f luorescence tivation period. M represents the difference between C-capture determination (Doan and Obbard 2011). The dye solu- the mass of carbon integrated into the microalgal biomass tion (100 μL) was then added to 1 mL algal suspension, by photosynthesis and the mass of carbon released by vortexed for 10 s, and incubated at ambient temperature microalgal and bacterial cells through aerobic respiration. for 5 min to make sure the stain went into the cells. After M > 0 indicates a net carbon capture via photosyn- C-capture being thoroughly vortexed, the f luorescence intensity of thesis, while M < 0 indicates a net carbon release by C-capture the suspension was analysed using CytoFLEX LX flow aerobic respiration. cytometer. Fluorescence measurements were carried out 1 3 Journal of Applied Phycology Statistical analysis Data in this study are expressed as mean ± standard devia- tion (SD) of a total of four measurements (2 biological rep- licates × 2 analytical replicates). The statistical analysis was carried out using SPSS Statistics (version 27.0.1, IBM). Statistical significance (p < 0.05) between the results of selected cultures was assessed by one-way analysis of vari- ance (ANOVA) using Post Hoc Tukey’s test. Results Capacity to grow under mixotrophic or heterotrophic conditions The actual final biomass concentration, maximum biomass concentration, maximum specific growth rate and lag time Fig. 1 Growth curve of N. oceanica under autotrophic, mixotrophic for each cultivation are shown in Fig. 1 and Table 1. Nan- (M), heterotrophic (H) and dark-control cultivations nochloropsis oceanica cultivated mixotrophically with 1% (w/v) initial lactose concentration presented the highest final −1 be able to assimilate lactose under mixotrophic rather than biomass concentration of 0.71 g L at the end of cultivation heterotrophic conditions. period, followed by the culture with 0.5% (w/v) initial lac- −1 tose concentration at 0.56 ± 0.01 g L in mixotrophic group. Algae‑bacteria population dynamics This biomass concentration was 4 – 4.5 higher than those obtained in the heterotrophic group, which experienced only −1 Flow cytometry with DAPI nucleus fluorescence staining modest increases in biomass concentration (ca. 0.15 g L was used to identify the population dynamics in algal sus- regardless of the lactose concentration). The dark-control pensions. Both microalgae cells and bacterial cells were group understandably experienced almost no growth given stained by DAPI, while only the microalgae cells emitted that it lacked both light for photosynthesis and organic car- autofluorescence. Microalgal cell concentration was there- bon supply. Previous studies (Espinosa-Gonzalez et al. 2014; fore quantified based on autofluorescence cell count. In the Hemalatha et al. 2019; Zanette et al. 2019) have reported mixotrophic cultures, algal cell count displayed the same a higher final biomass concentration for mixotrophic con - increasing pattern as biomass concentration. Both FSC ditions, which could be probably attributed to higher light median and SSC median also showed a significant increase intensity and more effective agitation and aeration systems. by the end of cultivation (Fig. 2a and b). The shift of FSC Nevertheless, it was obvious that lactose was able to enhance median in the mixotrophic culture with 1% (w/v) initial lac- algal growth relative to autotrophic system, and the mixo- tose concentration can be observed in Fig. 2d. These results trophic cultivation using lactose showed a strong potential indicated that both microalgae cell concentration and cell to obtain high biomass concentration. Overall, the results size increased during the mixotrophic cultivation, and that suggested a photosynthetic dependence of lactose assimi- the presence of lactose resulted in a positive impact on algae lation in N. oceanica, where the species appeared to only Table 1 The final biomass −1 −1 −1 Y (g L ) Y (g L ) (day ) (days) Algal cell count actual max max −1 concentration and growth (cells mL ) kinetics of N. oceanica under Autotrophic 0.46 ± 0.01 0.46 ± 0.00 0.08 ± 0.00 6.09 ± 0.13 1.65 × 10 autotrophic, mixotrophic (M) and heterotrophic (H) 1% Lactose (M) 0.71 ± 0.00 0.79 ± 0.02 0.10 ± 0.00 5.91 ± 0.18 2.00 × 10 cultivations 0.5% Lactose (M) 0.56 ± 0.01 0.65 ± 0.03 0.07 ± 0.00 5.93 ± 0.65 1.85 × 10 Dark-control 0.05 ± 0.00 - - - 4.71 × 10 1% Lactose (H) 0.16 ± 0.01 0.16 ± 0.00 0.13 ± 0.01 12.00 ± 0.10 5.88 × 10 0.5% Lactose (H) 0.14 ± 0.01 - - - 5.54 × 10 “-” means curve cannot be fitted using the growth model 1 3 Journal of Applied Phycology Fig. 2 Changes of algal cell population dynamics in the cultures dur- ▸ ing cultivation based on flow-cytometry analysis. (a) FSC median, (b) SSC median, (c) algal cell counts, and (d) changes of FSC median in mixotrophic culture with 1% (w/v) initial lactose concentration during cultivation growth, which was in agreement with not only biomass con- centration results presented in previous section based on OD measurements but also other studies investigating lactose assimilation in microalgae (Choi et al. 2018; Suwal et al. 2019; Zanette et al. 2019). On the other hand, a reduction in microalgae cell counts could be observed in the heterotrophic cultures (Fig. 2c). This was in contrast to the results obtained in previous section which showed a modest increase in biomass con- centration in the heterotrophic cultures. This discrepancy can likely be attributed to the proliferation of another set of microbial populations (i.e., bacteria) under heterotrophic conditions which contributed to the OD increase in the culture and thus led to the erroneous impression of algae growth in the heterotrophic system. The flow cytometer results were hence further processed to quantify the level of bacteria in the culture. The concentration of bacterial cells could not be directly quantified by flow cytometry under the current configurations due to the fact that bacterial cells did not emit autofluorescence and DAPI staining alone led to a high background noise that prevented quantification by particle (or cell) counts. Instead, we indirectly deduced bac- terial mass contribution in the culture by using autofluores- cence measurement of algal cells (from flow cytometry) as the basis of microalgae contribution to the overall biomass concentration in the culture and subtracting such contribu- tion from the measurement of total biomass concentration to determine the mass proportion of bacteria in the culture. Equation 4 normalised the algal cell count in the culture (e.g. mixotrophic or heterotrophic systems) to that obtained in the autotrophic system based on autofluorescence flow cytometry and used the resulting ratio to calculate the algae biomass concentration in the culture. The bacterial biomass concentration was then calculated by subtracting the value of algae biomass concentration from the overall biomass concentration in the culture (Eq. 5). As shown in Fig. 3, the increase in the population of associated bacteria was observed in both mixotrophic and heterotrophic cultures. Cultures grown heterotrophically, however, showed sig- nificantly higher bacteria concentration after 3 weeks of −1 cultivation, reaching 0.14 – 0.15 g L in concentration. Bacteria emerged as the dominant population in the cul- tures, occupying ca. 96% of total biomass in heterotrophic cultures. The biomass concentration increase observed in the heterotrophic culture (previous section) can therefore almost entirely be attributed to the proliferation of bacteria rather than microalgae growth. 1 3 Journal of Applied Phycology Fig. 3 Changes in microalgae and associated-bacteria biomass concentration during cultiva- tion (top) and the proportion of microalgae and bacteria at the end of cultivation periods (bottom). M: Mixotrophy, H: Heterotrophy The analysis in Fig. 3 also revealed that bacteria made greater than those obtained under heterotrophic conditions 5 −1 up only < 12% of biomass in mixotrophic cultures and that (ca. 6.00 × 10 cells mL ) (Table 1). The results are well microalgae cells were able to suppress bacterial growth and aligned with biomass concentration measurements in previ- maintain dominance in these cultures. The algal cell counts ous section showing significantly higher biomass growth in attained by the mixotrophic cultures (1.85 × 10 to 2.00 × the mixotrophic cultures than in the heterotrophic cultures. 7 −1 10 cells mL ) after 30-day cultivation were ca. 35-fold N. oceanica thus appeared to be able to utilize lactose only 1 3 Journal of Applied Phycology Table 2 Reduction of nitrate, phosphate and lactose in the medium by the end of cultivation period − − Lactose (%) NO (%) PO (%) 3 4 Autotrophic N/A 100 ± 0.00 96.63 ± 0.02 1% Lactose (M) 56.67 ± 0.85 100 ± 0.00 95.54 ± 0.25 0.5% Lactose (M) 37.41 ± 1.99 100 ± 0.00 95.05 ± 0.01 Dark-control N/A 59.32 ± 0.62 92.70 ± 0.43 1% Lactose (H) 12.83 ± 0.56 100 ± 0.00 93.76 ± 0.49 0.5% Lactose (H) 8.18 ± 1.04 100 ± 0.00 93.73 ± 0.41 N/A Not available when cultivated in the presence of light. In darkness, the lactose assimilation pathways were suppressed, allowing native bacteria present in the culture to proliferate and gain dominance instead. Lactose assimilation The reduction of lactose and other nutrients in culture medium at the end of cultivation cycle is shown in Table 2. Lactose metabo- lization was observed in both 1% (w/v) and 0.5% (w/v) mixo- trophic cultures, which showed 57.2% and 37.4% reduction in lactose concentration by day 30, respectively (Table 2, Fig. 4c). −1 Glucose and galactose concentrations remained below 0.1 g L at all time points across all mixotrophic cultures, which was in agreement with the study of Zanette et al. (2019). The results supported our findings in previous sections and confirmed that the cells in N. oceanica cultures were able to assimilate lactose under mixotrophic conditions. Despite this, the pathways which the cells utilized to assimilate lactose is currently unknown. We speculate that the cells can either absorb lactose directly from the medium and then break the disaccharides down to glucose and galactose using intracellular β-galactosidase or they can release extracellular β-galactosidase to hydrolyse lactose in the medium into glucose and galactose before subsequently metabolising the resulting monosaccharides or undergo a combination of the two pathways. Assaying the medium for extracellular β-galactosidase activities can provide further insights into lactose assimilation. Regardless, the fact that there was no monosaccharide accu- mulation observed at any time point across both mixotrophic cultures was suggestive of the fact that Nannochloropsis cells had the capacity to indiscriminately assimilate both glucose and galactose molecules derived from lactose hydrolysis (Girard et al. 2014). On the other hand, the heterotrophic cultures experienced minimum lactose reduction (ca. < 10% reduction). This sup- ported the initial observation in the previous sections that N. oceanica were unable to activate lactose assimilation path- ways in darkness. Interestingly, since the heterotrophic cul- Fig. 4 Changes in the concentrations of lactose (a), nitrate (b) and phosphate (c) during cultivation. M: Mixotrophy, H: Heterotrophy tures have been overtaken by native bacteria (as evidenced 1 3 Journal of Applied Phycology The subsequent decrease in lactase activity (day 8 – 16) cor- responded to the mid-point in the exponential growth phase where the cells began to experience declining growth rate in anticipation of nitrogen depletion (~ day 12) and the ces- sation of cell division. The results were in agreement with the study conducted by Bentahar et al. (2019b), indicating a higher enzyme productions in their mixotrophic cultures of T. obliquus after 8 cultivation days and that biomass pro- ductivity and enzyme productions were related to the trophic stage. Moreover, a similar behaviour could be observed in yeast cells. Studies conducted by Branco et al. (2020) and Chniti et al. (2017) indicated that young and budding yeast cells showed a higher metabolic rate on sugar consumption for producing ethanol, while maturated cells only consumed smaller amounts of sugar for maintenance. Through these findings, this study showed that cultivation time played an important role in regulating β-galactosidase activity in N. oceanica. Future industrial application planning to use the Fig. 5 Extracellular β-galactosidase production during mixotrophic species for the production of β-galactosidase enzyme will (M) and heterotrophic (H) cultivations thus have to optimise their harvest time (just before the start of stationary phase) to target maximum enzyme activity. by the flow cytometry results in previous section), the mini- In contrast, no extracellular β-galactosidase production mum lactose utilisation observed in these cultures would was observed in heterotrophic cultures, thus validating our suggest that these bacterial cells also lacked the ability to initial observation that a) N. oceanica were unable to acti- effectively metabolise lactose. vate lactose assimilation pathways in darkness and b) the native bacteria that have emerged as dominant population β‑Galactosidase production and kinetics in the culture also lacked the ability to produce extracel- lular β-galactosidase. The absence of β-galactosidase in In order to better understand the underlying pathways associ- the bacteria-dominated heterotrophic cultures served to ated with lactose assimilation in N. oceanica cultures, extra- reinforce the endogenous nature of enzyme production by cellular β-galactosidase activity in the medium was assayed microalgae in the mixotrophic cultures, ruling out the pos- every four days. The ability of N. oceanica to produce extra- sibility that the β-galactosidase detected in the mixotrophic cellular β-galactosidase to process lactose when cultivated medium was derived from bacteria cells or microalgae-bac- under mixotrophic conditions were demonstrated in Fig. 5, teria interactions. with the highest specific enzymatic activity found in cul- tures with 0.5% (w/v) initial lactose concentration on day Removal of other nutrients −1 8 at 41.47 ± 0.33 U g . The change in specific enzy - biomass matic activity in mixotrophic cultures over time followed Nitrate and phosphate removals were studied to understand the same pattern in both mixotrophic experiments, showing the effect of lactose assimilation on bioremediation perfor - a rapid rise at the early stage of cultivation before reaching mance. Apart from the dark-control group, all of the other a peak on day 8 and experiencing a sharp decline to day cultures were able to completely deplete nitrate and remove 14. A low level of β-galactosidase activity persisted in the 94 – 97% of phosphate in their medium, regardless of auto- −1 last two weeks of the cultivation (less than 0.5 U g trophy, mixotrophy or heterotrophy (Fig. 4b, c, Table 2). The biomass on day 22). Similar findings were reported by Suwal et al. rate of nitrate and phosphate removal appeared identical for (2019), who observed β-galactosidase activity in Tetrades- both mixotrophic and autotrophic cultures, despite the higher mus obliquus when treating whey permeate to reach a pla- growth rates observed in the mixotrophic cultures, indicating teau before decreasing by half at the end of cultivation. The that the cultures were likely nitrate- and phosphate- limited kinetics of β-galactosidase activity observed in present study (Halim et al. 2019b). In comparison with the nitrate removal could be attributed to the different stages in the microalgae in mixotrophic cultures, nitrate content was reduced more growth cycle (Fig. 1). The initial increase in activity (day slowly in the heterotrophic culture and dark-control group. 4 – 8) can be correlated to the transition from lag phase to This was expected as these cultures exhibited significantly exponential phase where cells began to undergo rapid cell lower rate of biomass concentration increase relative to their division and were hungry for ‘carbon’ to support growth. light-experiment counterparts. Since bacteria have overtaken 1 3 Journal of Applied Phycology microalgae as dominant population in the heterotrophic cul- studies investigating mixotrophy in microalgae (Smith et al. tures, the reduction in nitrate and phosphate concentration in 2015; Zhang et al. 2021) and can be attributed to the low these cultures can likely be ascribed to bacterial utilization concentration of atmospheric C O present in the air (0.04%) (rather than microalgal assimilation). Lactose, nitrate and and the associated limitation of diffusive mass transfer into phosphate are common components of dairy side streams; aqueous solution. the high removal efficiency for these nutrients as reported In the heterotrophic cultures the dominant carbon metab- in this study alludes to the promising nature of N. oceanica olism was aerobic respiration by both microalgae (due to the mixotrophic cultivation as a promising green strategy for lack of algal photosynthesis) and associated bacteria leading valorisation of dairy side streams. to a net CO release. The inability of N. oceanica and asso- ciated bacteria to activate extracellular lactose assimilation Carbon balance in the dark, however, meant that there was limited biomass growth and thus minimal net CO evolution in the hetero- −1 To further probe microalgal carbon metabolism during mix- trophic cultures (between 0.07 – 0.40 g L ). otrophic and heterotrophic cultivation, carbon mass balance was carried out. The total mass of carbon stored in algal biomass, the mass of carbon assimilated from lactose and Discussion the net mass of carbon released or consumed as atmospheric carbon dioxide were shown in Table 3. A net carbon seques- Findings from previous sections enabled us to develop a −1 tration of 0.23 ± 0.00 g L was observed in autotrophic working model for N. oceanica lactose assimilation and cultures. This was expected as the accumulation of carbon in β-galactosidase production under both mixotrophic and algal biomass in autotrophic cultivation was solely depend- heterotrophic conditions. Under mixotrophic conditions, ent on the difference between CO fixation during photosyn- the cells were able to activate lactose assimilation pathways thesis and respiratory C O release. In contrast to autotrophic where they secreted extracellular β-galactosidase into the cultures, carbon flow in the other regimes (i.e., dark-control, medium to hydrolyse lactose (up to 57% of available lac- mixotrophic and heterotrophic cultivations) resulted in a net tose) into glucose and galactose. β-Galactosidase production CO release rather than CO fixation. The highest net CO reached a peak at mid-logarithmic phase (day 8 of cultiva- 2 2 2 −1 release was found in mixotrophic culture at 1% (w/v) initial tion at 41.47 ± 0.33 U g ) before experiencing sig- biomass −1 lactose concentration (1.75 ± 0.07 g L ). The apparent net nificant decline, indicating that future industrial application CO release in mixotrophic cultures indicated that, in the planning to use N. oceanica for β-galactosidase synthesis presence of lactose, N. oceanica preferred to obtain carbon should aim for a short growth cycle. The cells then rap- through lactose assimilation rather than photosynthesis. idly absorbed the resulting glucose and galactose sub-units, This reduced the amount of carbon sequestered via photo- likely integrating both monosaccharides into standard gly- synthesis and led to the rate of carbon released by aerobic colytic pathways and tricarboxylic acid (TCA) cycle to fuel respiration to exceed that sequestered by photosynthesis. further energy and biomass generation. No accumulation The preferential uptake of dissolved organic carbon over of either glucose or galactose was observed in the medium, inorganic carbon has also previously been reported in other indicating that the cells were able to indiscriminately uptake both monosaccharides. The metabolization of lactose led to rapid microalgal cell division which depleted all available Table 3 The mass of carbon involved in biomass production and lactose nitrates and > 95% available phosphates in the medium and assimilation. The difference between mass of carbon assimilated into the resulted in a homogenous culture where microalgae cells biomass by photosynthesis and mass of carbon released by the biomass remained dominant (> 88% of total biomass). The mixo- due to respiration is calculated using Eq. 8 trophic pathways however came at the expense of photo- ** M M M C-biomass C-lactose C-capture −1 −1 −1 synthetic efficiency, resulting in a net release of CO to the (g L ) (g L ) (g L ) 2 C C C atmosphere. Autotrophic 0.23 ± 0.00 N/A 0.23 ± 0.00 Lactose assimilation in N. oceanica was found to be mix- 1% Lactose (M) 0.36 ± 0.00 2.11 ± 0.07 -1.75 ± 0.07 otrophic in nature, with the species being unable to secrete 0.5% Lactose (M) 0.28 ± 0.00 0.73 ± 0.06 -0.45 ± 0.06 any β-galactosidase into the medium under heterotrophic Dark-control 0.03 ± 0.00 N/A -0.03 ± 0.00 conditions. Heterotrophic conditions led to a decline in the 1% Lactose (H) 0.08 ± 0.01 0.48 ± 0.02 -0.40 ± 0.02 number of microalgae cells, in turn allowing native bacterial 0.5% Lactose (H) 0.07 ± 0.01 0.15 ± 0.04 -0.07 ± 0.04 cells to take over the culture and emerge as the dominant population (over 95% of total biomass). Interestingly, the N/A Not available ** native bacterial population also lacked the ability to secrete Positive and negative value represents net carbon fixation and net extracellular β-galactosidase and assimilate lactose in CO release, respectively 1 3 Journal of Applied Phycology darkness, as evidenced by the absence of the enzyme in the Nannochloropsis cultures had denitrifying capabilities, medium. Heterotrophic cultures metabolised less than 13% being able to metabolise organic carbon through the utilisa- of available lactose and thus experienced minimal overall tion of nitrate and oxygen as terminal electron acceptors biomass growth (final biomass concentration = 0.14 – 0.16 (Robertson and Gijs Kuenen 1984; Liu et al. 2016; Zhang −1 g L ) that was ca. four-fold lower than in the mixo- et al. 2016). biomass −1 trophic cultures (0.56 – 0.71 g L ). Despite this slow In terms of industrial implications, the results from this biomass growth, the cultures were still able to remove all available study highlighted the inherent limitations of using N. oce- nitrates and ca. 94% of available phosphates in the medium, anica to bioremediate lactose-rich dairy side streams while likely attributed to the denitrifying capacity of the native co-producing valuable β-galactosidase and EPA. The spe- bacteria typically isolated in Nannochloropsis culture (Pod- cies’ inability to utilize lactose under heterotrophic condi- dar et al. 2018). tions limits its application in treating carbon-rich dairy waste The underlying reason for the light-dependent nature to strict mixotrophic strategy. In the context of wastewater of lactose assimilation in Nannochloropsis is not well treatment in Europe, however, mixotrophic cultivation is understood but can likely be ascribed to the interde- less scalable than heterotrophic systems due to the need for pendence enzyme synthesis and adenosine triphosphate a) light irradiation (a limited natural resource in northern (ATP) availability. Photosynthesis provides substrates Europe) which leads to increased cost in artificial light- that are metabolized by algal cells to synthesise energy ing, b) larger land area to house photobioreactors for which (i.e., ATP) via aerobic respiration, thus driving other pro- many landlocked food operators (e.g., dairy processors) sim- cesses in living cells such as cellular maintenance and ply lack the space to accommodate. Photobioreactors for chemical/enzyme synthesis, including those needed for autotrophic and mixotrophic cultivation are also generally the assimilation of external nutrients (Knowles 1980). considered to more expensive to build and operate than fer- While in heterotrophic cultivation, no additional oxy- menters used for heterotrophic growth. Open-raceway ponds gen was generated due to the lack of photosynthesis for mixotrophic cultivation is not a viable option due to the and only the dissolved oxygen in the culture could be increased risk of bacterial contamination and culture crash. utilized for aerobic respiration. This likely resulted in a Future research effort should focus on screening Nannochlo- limited energy production; cellular maintenance for basal ropsis species able to grow heterotrophically in lactose-rich metabolism therefore took priority over the production of dairy side-streams or further optimisation of mixotrophic non-essential enzymes, such as the production of extra- conditions to reduce light and heating requirements. cellular β-galactosidase, leading to undetectable level of enzymatic activity in heterotrophic cultures. The maximum microalgal enzymatic activity (41.47 ± 0.33 Conclusion −1 U g ) measured for mixotrophic cultures in this study biomass was lower than those previously reported in other studies This study investigated the mechanism of lactose assimila- with bacteria and yeasts (Carević et al. 2015; You et al. tion in N. oceanica for dairy-wastewater bioremediation and 2017). This can likely be ascribed to species variation and co-production of β-galactosidase. Lactose assimilation in N. specific cultivation conditions (e.g., temperature, pH and oceanica was found to require mixotrophic conditions, with medium composition). Previous studies have reported that biomass production in mixotrophic cultures reaching > four- ion composition in the medium can affect the activity of fold that in the heterotrophic systems. Under mixotrophic lactase derived from microorganisms (Jurado et al. 2004; conditions, maximum extracellular β-galactosidase secretion −1 Otieno 2010). In the present study, since artificial sea water (41.47 ± 0.33 U g ) was attained during mid-loga- biomass was employed as medium, the mineral ion concentrations rithmic phase when cells were still rapidly dividing. The were higher than those of freshwater medium and can have algal cells were able to indiscriminately absorb both glu- an inhibitory effect on the β-galactosidase activity. cose or galactose sub-units derived from lactose hydrolysis The identity of native bacteria associated with N. oce- and no monosaccharide accumulation was observed in the anica cultures was not investigated. However, other studies medium. Bacterial growth was supressed, and microalgae have shown that bacteria belonging to the Proteobacteria cells maintained dominance throughout cultivation (bacteria phylum to be dominant in Nannochloropsis sp. cultures accounting for < 12% of biomass by the end of cultivation). (Nakase and Eguchi 2007). Handley and Lloyd (2013) and Moreover, the mixotrophic system investigated in the study Kelly et al. (2006) have also indicated that Marinobacter has the potential to transform the carbon present in lactose- sp. and Paracoccus sp. were ubiquitous in marine environ- rich dairy wastewater into both microalgae biomass and C O ment as they possess the ability to resist high salinity and via respiration, thereby reducing the net amount of carbon low nutrient conditions. Poddar et al. (2018) have indicated expelled into the environment relative to the initial carbon that native Marinobacter alkaliphilus isolated from their in the wastewater. 1 3 Journal of Applied Phycology On the other hand, under heterotrophic conditions lac- References tose assimilation pathways in N. oceanica were inactivated Bentahar J, Deschênes JS (2022) Influence of sweet whey permeate resulting in native bacteria taking over the culture and occu- utilization on Tetradesmus obliquus growth and β-galactosidase pying over 95% of total biomass by the end of cultivation. production. Can J Chem Eng 100:1479–1488 The bacterial population, however, also appeared to lack the Bentahar J, Doyen A, Beaulieu L, Deschênes JS (2019a) Acid whey per- metabolic pathways needed to effectively assimilate lactose meate: An alternative growth medium for microalgae Tetradesmus obliquus and production of β-galactosidase. Algal Res 41:101559 in darkness, producing negligible amount of extracellular Bentahar J, Doyen A, Beaulieu L, Deschênes JS (2019b) Investigation β-galactosidase and consuming < 10% of available lactose. of β-galactosidase production by microalga Tetradesmus obliquus Results from the study indicate that N. oceanica can be effec- in determined growth conditions. J Appl Phycol 31:301–308 tively applied for onsite dairy wastewater treatment under Biswas T, Bhushan S, Prajapati SK, Ray Chaudhuri S (2021) An eco- friendly strategy for dairy wastewater remediation with high strict mixotrophic conditions. Its inability to assimilate lac- lipid microalgae-bacterial biomass production. J Environ Man- tose under heterotrophic conditions is commercially disad- age 286:112196 vantageous as it increases areal footprint and bioreactor costs. Branco RHR, Amândio MST, Serafim LS, Xavier AMRB (2020) Etha- nol production from hydrolyzed Kraft pulp by mono- and co-cul- tures of yeasts: The challenge of C6 and C5 sugars consumption. Authors’ contributions Yuchen Li: Conceptualization, Methodology, Energies 13:en13030744 Validation, Formal analysis, Investigation, Writing; Svitlana Miros: Carević M, Vukašinović-Sekulić M, Grbavčić S, Stojanović M, Formal analysis, Writing, Review & editing; Hossein Kiani: Method- Mihailović M, Dimitrijević A, Bezbradica D (2015) Optimization ology, Investigation; Hans-Georg Eckhardt: Formal analysis, Review of β-galactosidase production from lactic acid bacteria. Hemijska & editing; Alfonso Blanco: Formal analysis, Review & editing; Shane Industrija 69:305–312 Mulcahy: Review & editing; Hugh McDonnell: Review & editing, Chen WH, Lin BJ, Huang MY, Chang JS (2015) Thermochemical con- Funding acquisition; Brijesh Kumar Tiwari: Resources, Review & version of microalgal biomass into biofuels: A review. Bioresour editing; Ronald Halim: Conceptualization, Methodology, Resources, Technol 184:314–327 Project administration, Review & editing, Funding acquisition. Chniti S, Jemni M, Bentaha I et al (2017) Kinetic of sugar consump- tion and ethanol production on very high gravity fermentation Funding Open Access funding provided by the IReL Consor- from syrup of dates by-products (Phoenix dactylifera L.) by using tium Yuchen Li would like to acknowledge the University Col- Saccharomyces cerevisiae, Candida pelliculosa and Zygosaccha- lege Dublin-China Scholarship Council (UCD-CSC) Programme romyces rouxii. J Microbiol Biotechnol Food Sci 7:199–203 for their financial support. Svitlana Miros would like to acknowl- Choi YK, Jang HM, Kan E (2018) Microalgal biomass and lipid pro- edge the Irish Research Council Ukrainian Researchers Scheme duction on dairy effluent using a novel microalga, Chlorella sp. (URS/2022/3L) for their financial support. Hossein Kiani would isolated from dairy wastewater. Biotech Bioproc Eng 23:333–340 like to acknowledge Enterprise Ireland and the European Union’s Correia DM, Dias LG, Veloso ACA, Dias T, Rocha I, Rodrigues Horizon 2020 Research and innovation Programme under the Marie LR, Peres AM (2014) Dietary sugars analysis: quantification Skłodowska-Curie Co-funding of regional, national and interna- of fructooligosacharides during fermentation by HPLC-RI tional programmes (Project ID: MF 2020 0108) for their financial method. Front Nutr 1:11 support. da Silva TL, Moniz P, Silva C, Reis A (2021) The role of heterotrophic microalgae in waste conversion to biofuels and bioproducts. Pro- Data availability The raw data supporting the conclusions of this arti- cesses 9:1090 cle will be made available by the authors to any qualified researcher Daneshvar E, Zarrinmehr MJ, Hashtjin AM, Farhadian O, Bhatnagar upon request. A (2018) Versatile applications of freshwater and marine water microalgae in dairy wastewater treatment, lipid extraction and tetracycline biosorption. Bioresour Technol 268:523–530 Declarations Daneshvar E, Zarrinmehr MJ, Koutra E, Kornaros M, Farhadian O, Bhat- nagar A (2019) Sequential cultivation of microalgae in raw and recy- Competing interests The authors declare that they have no competing cled dairy wastewater: Microalgal growth, wastewater treatment and financial interests or personal relationships that could have appeared biochemical composition. Bioresour Technol 273:556–564 to influence the work reported in this paper. Doan TTY, Obbard JP (2011) Improved Nile Red staining of Nanno- chloropsis sp. J Appl Phycol 23:895–901 Open Access This article is licensed under a Creative Commons Attri- Espinosa-Gonzalez I, Parashar A, Bressler DC (2014) Heterotrophic bution 4.0 International License, which permits use, sharing, adapta- growth and lipid accumulation of Chlorella protothecoides in tion, distribution and reproduction in any medium or format, as long whey permeate, a dairy by-product stream, for biofuel produc- as you give appropriate credit to the original author(s) and the source, tion. Bioresour Technol 155:170–176 provide a link to the Creative Commons licence, and indicate if changes Girard J-M, Roy M-L, Hafsa MB, Gagnon J, Faucheux N, Heitz M, were made. The images or other third party material in this article are Tremblay R, Deschênes J-S (2014) Mixotrophic cultivation of included in the article's Creative Commons licence, unless indicated green microalgae Scenedesmus obliquus on cheese whey perme- otherwise in a credit line to the material. If material is not included in ate for biodiesel production. Algal Res 5:241–248 the article's Creative Commons licence and your intended use is not Gramegna G, Scortica A, Scafati V, Ferella F, Gurrieri L, Giovannoni permitted by statutory regulation or exceeds the permitted use, you will M, Bassi R, Sparla F, Mattei B, Benedetti M et al (2020) Explor- need to obtain permission directly from the copyright holder. To view a ing the potential of microalgae in the recycling of dairy wastes. copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . Bioresour Technol Rep 12:100604 1 3 Journal of Applied Phycology Guillard RRL, Ryther JH (1962) Studies of marine planktonic diatoms. Poliner E, Pulman JA, Zienkiewicz K, Childs K, Benning C, Farré I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) EM (2018) A toolkit for Nannochloropsis oceanica CCMP1779 Gran. Can J Microbiol 8:229–239 enables gene stacking and genetic engineering of the eicosapen- Halim R, Hill DRA, Hanssen E, Webley PA, Blackburn S, Gross- taenoic acid pathway for enhanced long-chain polyunsaturated man AR, Posten C, Martin GJO (2019a) Towards sustainable fatty acid production. Plant Biotechnol J 16:298–309 microalgal biomass processing: Anaerobic induction of autol- Robertson LA, Kuenen JG (1984) Aerobic denitrification: a contro- ytic cell-wall self-ingestion in lipid-rich: Nannochloropsis slur- versy revived. Arch Microbiol 139:351–354 ries. Green Chem 21:2967–2982 Sahin D, Altindag UH, Tas E (2018) Enhancement of docosahexaenoic Halim R, Hill DRA, Hanssen E, Webley PA, Martin GJO (2019b) acid (DHA) and beta-carotene production in Schizochytrium sp. Thermally coupled dark-anoxia incubation: A platform technology using symbiotic relationship with Rhodotorula glutinis. Process to induce auto-fermentation and thus cell-wall thinning in both Biochem 75:10–15 nitrogen-replete and nitrogen-deplete Nannochloropsis slurries. Slavov AK (2017) General characteristics and treatment possibilities Bioresour Technol 290:121769 of dairy wastewater -a review. Food Technol Biotechnol 55:14–28 Handley KM, Lloyd JR (2013) Biogeochemical implications of the Smith RT, Bangert K, Wilkinson SJ, Gilmour DJ (2015) Synergis- ubiquitous colonization of marine habitats and redox gradients tic carbon metabolism in a fast growing mixotrophic freshwater by Marinobacter species. Front Microbiol 4:136 microalgal species Micractinium inermum. Biomass Bioenergy Hemalatha M, Sravan JS, Min B, Venkata Mohan S (2019) Microalgae- 82:73–86 biorefinery with cascading resource recovery design associated Suwal S, Bentahar J, Marciniak A, Beaulieu L, Deschênes J-S, Doyen to dairy wastewater treatment. Bioresour Technol 284:424–429 A (2019) Evidence of the production of galactooligosaccharide Hulatt CJ, Wijffels RH, Bolla S, Kiron V (2017) Production of fatty from whey permeate by the microalgae Tetradesmus obliquus. acids and protein by Nannochloropsis in flat-plate photobioreac- Algal Res 39:101470 tors. PLoS One 12:e0170440 Tian-Yuan Z, Yin-Hu W, Lin-Lan Z, Xiao-Xiong W, Hong-Ying H Jurado E, Camacho F, Luzón G, Vicaria JM (2004) Kinetic models of (2014) Screening heterotrophic microalgal strains by using the activity for β-galactosidases: Influence of pH, ionic concentration Biolog method for biofuel production from organic wastewater. and temperature. Enzyme Microb Technol 34:33–40 Algal Res 6:175–179 Kelly DP, Rainey FA, Wood AP (2006) The genus Paracoccus. In: Yin FW, Zhu SY, Guo DS, Ren LJ, Ji XJ, Huang H, Gao Z (2019) Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt Development of a strategy for the production of docosahexaenoic E (eds) The Prokaryotes. Springer, New York, pp 232–249 acid by Schizochytrium sp. from cane molasses and algae-residue. Kiani H, Azimi Y, Li Y, Mousavi M, Cara F, Mulcahy S, McDonnell H, Bioresour Technol 271:118–124 Blanco A, Halim R (2023) Nitrogen and phosphate removal from You SP, Wang XN, Qi W, Sy RX, Hw ZM (2017) Optimisation of dairy processing side-streams by monocultures or consortium of culture conditions and development of a novel fed-batch strategy microalgae. J Biotechnol 361:1–11 for high production of β-galactosidase by Kluyveromyces lactis. Knowles JR (1980) Enzyme-catalyzed phosphoryl transfer reactions. Int J Food Sci Technol 52:1887–1893 Annu Rev Biochem 49:877–919 Zanette CM, Mariano AB, Yukawa YS, Mendes I, Spier MR (2019) Kolanowski W (2021) Salmonids as natural functional food rich in Microalgae mixotrophic cultivation for β-galactosidase produc- Omega-3 PUFA. Appl Sci 11:2409 tion. J Appl Phycol 31:1597–1606 Lawford HQ, Rousseau JD (1996) Studies on nutrient requirements and Zhang S, Pang S, Wang P, Wang C, Guo C, Addo FG, Li Y (2016) cost-effective supplements for ethanol production by recombinant Responses of bacterial community structure and denitrifying E. coli. Appl Biochem Biotech A 57–58:307–326 bacteria in biofilm to submerged macrophytes and nitrate. Sci Liu Y, Ai GM, Miao LL, Liu ZP (2016) Marinobacter strain NNA5, Rep 6:36178 a newly isolated and highly efficient aerobic denitrifier with zero Zhang Z, Sun D, Cheng KW, Chen F (2021) Investigation of carbon N O emission. Bioresour Technol 206:9–15 and energy metabolic mechanism of mixotrophy in Chromochloris Mapstone LJ, Taunt HN, Cui J, Purton S, Brooks TGR (2022) ADA: zofingiensis. Biotechnol Biofuels 14 an open-source software platform for plotting and analysis of data Zimermann JDaF, Sydney EB, Cerri ML, de Carvalho IK, Schafranski from laboratory photobioreactors. Appl Phycol 3:16–26 K, Sydney ACN, Vitali L, Gonçalves S, Micke GA, Soccol CR, Nakase G, Eguchi M (2007) Analysis of bacterial communities in Nannochloro- Demiate IM (2020) Growth kinetics, phenolic compounds profile psis sp. cultures used for larval fish production. Fisheries Sci 73:543–549 and pigments analysis of Galdieria sulphuraria cultivated in whey National Research Council (1981) Food Chemicals Codex, 3rd edn. permeate in shake-flasks and stirred-tank bioreactor. J Water Pro- The National Academies Press, Washington, DC cess Eng 38:101598 Otieno DO (2010) Synthesis of β-galactooligosaccharides from lactose using Zwietering MH, Jongenburger I, Rombouts FM, Van’t Riet K (1990) microbial β-galactosidases. Compr Rev Food Sci Food Saf 9:471–482 Modeling of the bacterial growth curve. Appl Environ Microbiol Poddar N, Sen R, Martin GJO (2018) Glycerol and nitrate utilisation by 56:1875–1881 marine microalgae Nannochloropsis salina and Chlorella sp. and associated bacteria during mixotrophic and heterotrophic growth. Publisher's note Springer Nature remains neutral with regard to Algal Res 33:298–309 jurisdictional claims in published maps and institutional affiliations. Półbrat T, Konkol D, Korczyński M (2021) Optimization of docosahex- aenoic acid production by Schizochytrium sp. – A review. Biocatal Agric Biotechnol 35:102076 1 3
Journal of Applied Phycology – Springer Journals
Published: Aug 1, 2023
Keywords: Lactose; β-galactosidase; Microalgae; Mixotrophy; Dairy, Nannochloropsis
You can share this free article with as many people as you like with the url below! We hope you enjoy this feature!
Read and print from thousands of top scholarly journals.
Already have an account? Log in
Bookmark this article. You can see your Bookmarks on your DeepDyve Library.
To save an article, log in first, or sign up for a DeepDyve account if you don’t already have one.
Copy and paste the desired citation format or use the link below to download a file formatted for EndNote
Access the full text.
Sign up today, get DeepDyve free for 14 days.
All DeepDyve websites use cookies to improve your online experience. They were placed on your computer when you launched this website. You can change your cookie settings through your browser.