In situ quantification of poly(3-hydroxybutyrate) and biomass in Cupriavidus necator by a fluorescence spectroscopic assay

Fluorescence spectroscopy offers a cheap, simple, and fast approach to monitor poly(3-hydroxybutyrate) (PHB) formation, a biodegradable polymer belonging to the biodegradable polyester class polyhydroxyalkanoates. In the present study, a fluorescence and side scatter-based spectroscopic setup was developed to monitor in situ biomass, and PHB formation of biotechnological applied Cupriavidus necator strain. To establish PHB quantification of C. necator, the dyes 2,2-difluoro-4,6,8,10,12-pentamethyl-3-aza-1-azonia-2-boranuidatricyclo[7.3.0.03,7]dodeca-1(12),4,6,8,10-pentaene (BODIPY493/503), ethyl 5-methoxy-1,2-bis(3-methylbut-2-enyl)-3-oxoindole-2-carboxylate (LipidGreen2), and 9-(diethylamino)benzo[a]phenoxazin-5-one (Nile red) were compared with each other. Fluorescence staining efficacy was obtained through 3D-excitation-emission matrix and design of experiments. The coefficients of determination were ≥ 0.98 for all three dyes and linear to the high-pressure liquid chromatography obtained PHB content, and the side scatter to the biomass concentration. The fluorescence correlation models were further improved by the incorporation of the biomass-related side scatter. Afterward, the resulting regression fluorescence models were successfully applied to nitrogen-deficit, phosphor-deficit, and NaCl-stressed C. necator cultures. The highest transferability of the regression models was shown by using LipidGreen2. The novel approach opens a tailor-made way for a fast and simultaneous detection of the crucial biotechnological parameters biomass and PHB content during fermentation. Key points • Intracellular quantification of PHB and biomass using fluorescence spectroscopy. • Optimizing fluorescence staining conditions and 3D-excitation-emission matrix. • PHB was best obtained by LipidGreen2, followed by BODIPDY493/503 and Nile red. Graphical abstract

Biogenic Sulfur-Based Chalcogenide Nanocrystals: Methods of Fabrication, Mechanistic Aspects, and Bio-Applications

Bio-nanotechnology has emerged as an efficient and competitive methodology for the production of added-value nanomaterials (NMs). This review article gathers knowledge gleaned from the literature regarding the biosynthesis of sulfur-based chalcogenide nanoparticles (S-NPs), such as CdS, ZnS and PbS NPs, using various biological resources, namely bacteria, fungi including yeast, algae, plant extracts, single biomolecules, and viruses. In addition, this work sheds light onto the hypothetical mechanistic aspects, and discusses the impact of varying the experimental parameters, such as the employed bio-entity, time, pH, and biomass concentration, on the obtained S-NPs and, consequently, on their properties. Furthermore, various bio-applications of these NMs are described. Finally, key elements regarding the whole process are summed up and some hints are provided to overcome encountered bottlenecks towards the improved and scalable production of biogenic S-NPs.

Assessment of Domestic Wastewaters as Potential Growth Media for Chlorella vulgaris and Haematococcus pluvialis

Domestic wastewater contains chemical compounds that can be used as nutrients for microalgae. Removing these chemical compounds from wastewater by microalgae might help in reducing the operation cost of wastewater management while minimizing the cultivation cost for large-scale microalgae metabolite production. In this study, domestic wastewater collected from Indah Water Konsortium (IWK), Kuala Lumpur, Malaysia, was assessed as growth media for two types of microalgae, namely Chlorella vulgaris and Haematococcus pluvialis. The biomass growth and nutrient removal efficiency of total nitrogen (TN), total phosphorus (TP), and total ammonia (TAN) in different concentrations of diluted wastewater were measured. The results showed that biomass concentration (0.227 g/L), biomass productivity (0.029 g/L/day), and specific growth rate (0,284 d-1) yielded by C. vulgaris in 14 days of 80% wastewater were comparable to those microalgae grew in standard Bold’s Basal medium (BBM). Besides, C. vulgaris grew in 50% wastewater to remove TN, TP, and TAN with the highest removal efficiency (>88%). For H. pluvialis, the biomass concentration in all wastewater concentrations was lower than BBM. The removal efficiencies of TN and TP were lower than 55%, but more than 80% for removal efficiency of TAN in 50% and 80% wastewater. Hence, C. vulgaris has better growth performance and nutrient removal efficiency than H. pluvialis. These findings indicated that IWK domestic wastewater could be used as growth media for microalgae, especially C. vulgaris.

Optical Detection of Harmful Algal Blooms in the Belgian Coastal Zone: A Cautionary Tale of Chlorophyll c3

Phaeocystis globosa is a nuisance haptophyte species that forms annual blooms in the southern North Sea and other coastal waters. At high biomass concentration, these are considered harmful algal blooms due to their deleterious impact on the local ecosystems and economy, and are considered an indicator for eutrophication. In the last two decades, methods have been developed for the optical detection and quantification of these blooms, with potential applications for autonomous in situ or remote observations. However, recent experimental evidence suggests that the interpretation of the optical signal and its exclusive association with P. globosa may not be accurate. In the North Sea, blooms of P. globosa are synchronous with those of the diatom Pseudo-nitzschia delicatissima, another harmful bloom-forming species with similar pigmentation and optical signature. Here we combine new and published measurements of pigmentation composition and inherent optical properties from pure cultures of several algal and cyanobacterial groups, together with environmental spectroscopy data, to identify the pigments generating the optical signals captured by two established algorithms. We further evaluate the association of those pigments and optical signals with P. globosa. We found that the interpretation of the pigment(s) generating the optical signals were incorrect and that previous methods are not specific to P. globosa, even in the context of the phytoplankton assemblage of the southern North Sea. Additionally, we found that the optical and pigment signatures of Phaeocystis species are part of a broad pigmentation trend across unrelated taxonomic groups related to chlorophyll c3 presence, with important consequences for the interpretation of pigment and optical data. We then develop and evaluate an algorithm to detect this pigmentation pattern with minimal influence of co-occurring species and elaborate general recommendations for the future development of algorithms.

Research technology of cultivation of spirulina algae with the use of CO2 hidden from the smoke of rice husk boiler

This paper presents an investigation of Spirulina algae cultivation by the CO2 gas emitted from the combustion of rice husk. The gas emitted from the rice husk combustion containing CO2 but no toxic gas of SOx. The CO2 molecules are absorbed into the micro-algae cultivation medium and then converted into the HCO3 by the assimilation of Spirulina. At the same time, the pH values are controlled to be from 8.5 to 9.5, which is suitable for Spirulina algae. At the first seven days of cultivation in Zarrouk medium the values of Spirulina algae biomass and pH increase from 0.05 g/l and 8.5 to 1.0 g/l and 10.2, respectively. On the 8th day, when the amount of 7,6 % CO2 v/v under 35–40 ºC and 1 atm is introduced into the above medium, the decrease of pH from 10.2 to 8.6 is observed. This pH value, which is maintained over the following days, is optimal for the growth yield of the Spirulina. As a result, the biomass concentration increases from 1.0 to 1.4 g/l. The obtained results are compared with those of the control sample from Zarrouk medium without gas introduction. For the latter case, the biomass reaches the maximum and then decreases. On the basis of the obtained results, the cultivation of Spirulina algae by using the CO2 molecules emitted from the combustion of rice husk can be applied practically.

Mass Transfer Features of Wavy-Bottomed Cascade Photobioreactors

It has been suggested that the energy-efficient production of microalgae biomass can be more easily obtained in short light path photobioreactors that can be operated at high biomass concentration. On the downside, however, high biomass concentrations also require an efficient gas exchange rate to avoid metabolic growth limitation or inhibition. A cascade photobioreactor featuring a thin liquid layer flowing down a sloping, wavy-bottomed surface can be operated at a biomass concentration that is much higher compared to most usual open-type equipment. Liquid flow, upon investigation, proved to exhibit peculiar “local recirculation” hydrodynamics, potentially conducive to the mixing of superficial and deep zones of the photobioreactor. Mass transfer coefficient represents a useful parameter to optimize the performance of a microalgal photobioreactor and its scale-up. The aim of the present article is to discuss the experimental mass transfer features of this novel type of photobioreactor and highlight expected opportunities and issues entailed by different ways of installing and operating such novel types of photobioreactors.

Submerged Fermentation of Animal Fat By-Products by Oleaginous Filamentous Fungi for the Production of Unsaturated Single Cell Oil

Animal waste fats were explored as a fermentation substrate for the production of high-value unsaturated single cell oil (SCO) using oleaginous fungi, Mucor circinelloides and Mortierella alpina. Both strains showed good growth and lipid accumulation when using animal fat as a single carbon source. The biomass concentration of 16.7 ± 2.2 gDCW/L and lipid content of 54.1%wt (of dry cell weight) were obtained for Mucor circinelloides in shake flask experiments, surpassing the biomass yield achieved in batch and fed-batch fermentation. In contrast, Mortierella alpina gave the highest biomass concentration (8.3 ± 0.3 gDCW/L) and lipid content (55.8%wt) in fed-batch fermentation. Fat grown Mortierella alpina was able to produce arachidonic acid (ARA), and the highest ARA content of 23.8%wt (of total lipid weight) was in fed-batch fermentation. Gamma-linolenic acid (GLA) was produced by both fungal strains. At the end of fed-batch fermentation, the GLA yields obtained for Mucor circinelloides and Mortierella alpina were 4.51%wt and 2.77%wt (of total lipid weight), respectively. This study demonstrates the production of unsaturated SCO-rich fungal biomass from animal fat by fermentation.

Control of Microalgae Growth in Artificially Lighted Photobioreactors Using Metaheuristic-Based Predictions

A metaheuristic algorithm can be a realistic solution when optimal control problems require a significant computational effort. The problem stated in this work concerns the optimal control of microalgae growth in an artificially lighted photobioreactor working in batch mode. The process and the dynamic model are very well known and have been validated in previous papers. The control solution is a closed-loop structure whose controller generates predicted control sequences. An efficient way to make optimal predictions is to use a metaheuristic algorithm, the particle swarm optimization algorithm. Even if this metaheuristic is efficient in treating predictions with a very large prediction horizon, the main objective of this paper is to find a tool to reduce the controller’s computational complexity. We propose a soft sensor that gives information used to reduce the interval where the control input’s values are placed in each sampling period. The sensor is based on measurement of the biomass concentration and numerical integration of the process model. The returned information concerns the specific growth rate of microalgae and the biomass yield on light energy. Algorithms, which can be used in real-time implementation, are proposed for all modules involved in the simulation series. Details concerning the implementation of the closed loop, controller, and soft sensor are presented. The simulation results prove that the soft sensor leads to a significant decrease in computational complexity.

Heterotrophically Ultrahigh-Cell-Density Cultivation of a High Protein-Yielding Unicellular Alga Chlorella With a Novel Nitrogen-Supply Strategy

The unicellular green alga Chlorella is an ideal protein source. However, the high production cost and low production capability of the current main photoautotrophic culture mode limit its application especially as an alternative protein source for food and feed, which might be overcome through high-cell-density cultivation in fermenters. In this study, a Chlorella sorokiniana strain CMBB276 with high protein content was selected from five Chlorella strains by comprehensive evaluation of their growth rates, protein contents, and yields. The optimal cultural temperature, pH, and mole ratio of carbon and nitrogen (C/N) for C. sorokiniana CMBB276 growth were found to be 30°C, 6.5, and 18, respectively. Ammonium chloride was proved to be the best nitrogen (N) source for C. sorokiniana CMBB276 growth, whereas growth inhibition caused by the accumulation of salts was observed under fed-batch cultivation when maintaining a constant C/N ratio of 18 by controlling pH with sodium hydroxide solution. By simultaneously reducing the concentration of ammonium chloride in the feeding medium and controlling pH with ammonium hydroxide, we finally achieved the ultrahigh-cell-density cultivation of C. sorokiniana CMBB276. The highest biomass concentration and protein yield reached 232 and 86.55 g l−1, respectively, showing the great potential of culturing C. sorokiniana CMBB276 in fermenters for economic and large-scale protein source production.

Filamentous Fungus Aspergillus oryzae for Food: From Submerged Cultivation to Fungal Burgers and Their Sensory Evaluation—A Pilot Study

New food sources are explored to provide food security in sustainable ways. The submerged fermentation of edible filamentous fungi is a promising strategy to provide nutritious and affordable food that is expected to have a low environmental impact. The aim of the current study was to assess the novel use of Aspergillus oryzae cultivated in submerged fermentation on oat flour as a source for food products that do not undergo secondary fermentation or significant downstream processing. The fungus was cultivated in a pilot-scale airlift bioreactor, and the biomass concentration and protein content of the biomass were assessed. A tasting with an untrained panel assessed consumer preferences regarding the taste and texture of minimally processed vegetarian and vegan burger patties made from the biomass, and how the patties fared against established meat-alternative-based patties. The cultivation of Aspergillus oryzae resulted in a yield of 6 g/L dry biomass with a protein content of 37% on a dry weight basis. The taste and texture of the minimally processed fungal burger patties were to the liking of some participants. This was also reflected in diverse feedback provided by the participants. The cultivation of the fungus on oat flour and its utilization in developing burger patties shows its promising potential for the production of nutritious food. The applications of the fungus can be further developed by exploring other favorable ways to texture and season this relatively new functional food source to the preferences of consumers.