Closed Cultivation System Research Articles

Analisis Kinetika Pertumbuhan Mikroalga Spirulina Sp. Menggunakan Pemodelan Richard dan Gompertz pada Fotobioreaktor Algaetree

Algaetree is a closed cultivation system with a photobioreactor type created by Gadjah Mada University in collaboration with the Indonesian Institute of Arts Yogyakarta. The aim of this research was to see the effectiveness of the growth kinetics of the microalgae type Spirulina sp. The research was carried out by carrying out variations in data sampling at 3 (three) points where microalgae grow in Algaetree. The dose was 10 ml using the sampling method, then 2 ml was taken to be examined using a microscope and the number of microalgae cells found on the haemocytometer was counted. The microalgae cells were studied and counted on 4 haemocytometer columns, then the number of cells was accumulated and the standard deviation and growth kinetics were calculated using Microsoft Excel and Python software. As a result, it was found that the highest cell number of microalgae on the Algaetree was 69166.67 cells/ml, with a standard deviation of 4249.18, while the maximum growth kinetics of microalgae on the Algaetree with the Richard model and the Gompertz model, the most significant value was the Richard model, namely (miumax ) 1.9958, curve fitting parameter (A) 6.8700, stationary phase (V) 397.43, and r-square (R^2) close to 1, namely 0.9674. This value shows that there is a match between the data processed and the cultivation system used. The results of the cultivation process can be seen that the kinetics of microalgae growth is quite significant, meaning that spirulina type microalgae can grow well in a tool called Algaetree. In the next process, Algaetree can be used as a reference object for comparing microalgae growth from a closed cultivation system with an open cultivation system.

Convolutional neural network regression for low-cost microalgal density estimation

Density of microalgae is critical information for production of algae in a closed cultivation system since it can be used to optimally control their growth rate, biomass concentration and quality of the products. Given advancement in image processing techniques and thanks to low-cost camera sensors, image based methods are increasingly widely utilized to indirectly estimate the density. Advantages of the image based techniques include being less invasive and more nondestructive and biosecured. However, most of the existing techniques rely on averaging all pixels of a microalgae image, which may eliminate crucial information of their spatial correlation. Therefore, in this work we propose to exploit a convolutional neural network (CNN) to efficiently extract information from the microalgae images, which are then employed to regress the density. Interestingly, the proposed deep CNN regression model accepts the whole color image as its input while the density is calculated in the output. The proposed CNN regression architecture was validated in real-world experiments where the microalgal strain Chlorella vulgaris was cultured and their images were captured by our low-cost camera sensor system. The obtained results demonstrate that the averaged estimation accuracy of the proposed model is 99.45% (± 0.68%) while the R2 value between the density predictions and the ground truths is 0.9997, which is highly accurate and practical.

Effect of excess CO2 on semi-continuous microalgae systems: Carbon biofixation

Microalgae-based CO2 fixation is a promising solution for achieving carbon neutrality. However, the low rate of carbon fixation is a significant issue due to gas transfer. A closed cultivation system was employed to overcome this challenge, incorporating headspace micro-positive pressure and elevated total CO2 (TCO2) levels to minimize CO2 loss and improve CO2 mass transfer. Microalgae could thrive and maintain a higher level of system pH within the TCO2 range of 0.12–0.49 mol CO2/L absorbent, and high TCO2 levels over 0.36 mol CO2/L absorbent were the optimal range. The semi-continuous microalgae system removed up to 8.72 ± 0.85 g CO2/L/day. Simultaneously, it exhibited phosphate and nitrate removal rates of 50.95 ± 10.71 and 182.14 ± 30.71 mg/L/day, respectively. The study found that 80% of the removed carbon was fixed as organic biomass. An increased level of TCO2 resulted in the accumulation of more lipids within the cells. Moreover, microalgae released carbon fixation products into the extracellular environment, and the dissolved organic carbon fixation rate reached as high as 14.58 ± 1.89 mmol/L/day. These findings suggest that microalgae use multiple pathways for carbon fixation, including biomass organic carbon accumulation, extracellular organic carbon secretion, and inorganic carbon precipitation. Improving carbon fixation rates may benefit the industrial application of microalgae technology, which aims to mitigate carbon emissions.

Improved Production of Astaxanthin from Haematococcus pluvialis Using a Hybrid Open–Closed Cultivation System

Haematococcus species are rich sources of the antioxidant astaxanthin and have good potential for carbon dioxide reduction. A variety of culture systems for these microalgae are currently in development, but clearly profitable approaches have yet to be reported. Open outdoor culture is currently the only feasible culture system for producing large amounts of biomass. In this study, based on laboratory results, the cultivation of Haematococcus was divided into two stages: a green stage characterised by cell growth, and a red stage characterised by astaxanthin accumulation. For mass culture, we adopted a hybrid open–closed pond system for astaxanthin production. The open culture system was shown to produce approximately 50 kg (dry weight) of biomass per culture at an average rate of 0.51 g L−1, with 0.52 μg mL−1 of astaxanthin content in a 12 -m3 water tank. As large amounts of microalgal bioproducts are in high demand, inexpensive open outdoor culture methods should be adopted as an alternative to costly closed photobioreactors. Although the levels of biomass and astaxanthin production were found to be 30% lower in the field than in the laboratory in this study, the basic data obtained in this research may be useful for lowering astaxanthin production costs.

Characterization of persistent marine bacterial community profiles isolated from long-term Kappaphycus alvarezii cultures in a closed cultivation system using 16S rDNA analysis

Characterization of persistent marine bacterial community profiles isolated from long-term Kappaphycus alvarezii cultures in a closed cultivation system using 16S rDNA analysis

Effects of citrate-stabilized gold and silver nanoparticles on some safety parameters of Porphyridium cruentum biomass.

Introduction: Our research raises the question of how realistic and safe it is to use gold and silver nanoparticles in biotechnologies to grow microalgae, which will later be used to obtain valuable products. To this purpose, it was necessary to assess the influence of 10 and 20nm Au and Ag nanoparticles stabilized in citrate on the growth of microalga Porphyridium cruentum in a closed cultivation system, as well as some safety parameters of biomass quality obtained under experimental conditions. Methods: Two types of experiments were conducted with the addition of nanoparticles on the first day and the fifth day of the cultivation cycle. Changes in productivity, lipid content, malondialdehyde (MDA), as well as antioxidant activity of microalgae biomass have been monitored in dynamics during the life cycle in a closed culture system. Results: The impact of nanoparticles on the growth curve of microalgae culture was marked by delaying the onset of the exponential growth phase. A significant increase in the content of lipids and MDA in biomass was noted. Excessive accumulation of lipid oxidation products within the first 24h of cultivation resulted in altered antioxidant activity of red algae extracts. Discussion: Citrate-stabilized gold and silver nanoparticles proved to be a stress factor for red microalga Porphyridium cruentum, causing significant changes in both biotechnological and biomass safety parameters. Addition of Au and Ag nanoparticles during the exponential growth phase of porphyridium culture led to an enhanced lipid accumulation and reduced MDA values in biomass.

Analysis of Microalgal Density Estimation by Using LASSO and Image Texture Features.

Monitoring and estimating the density of microalgae in a closed cultivation system is a critical task in culturing algae since it allows growers to optimally control both nutrients and cultivating conditions. Among the estimation techniques proposed so far, image-based methods, which are less invasive, nondestructive, and more biosecure, are practically preferred. Nevertheless, the premise behind most of those approaches is simply averaging the pixel values of images as inputs of a regression model to predict density values, which may not provide rich information of the microalgae presenting in the images. In this work, we propose to exploit more advanced texture features extracted from captured images, including confidence intervals of means of pixel values, powers of spatial frequencies presenting in images, and entropies accounting for pixel distribution. These diverse features can provide more information of microalgae, which can lead to more accurate estimation results. More importantly, we propose to use the texture features as inputs of a data-driven model based on L1 regularization, called least absolute shrinkage and selection operator (LASSO), where their coefficients are optimized in a manner that prioritizes more informative features. The LASSO model was then employed to efficiently estimate the density of microalgae presenting in a new image. The proposed approach was validated in real-world experiments monitoring the Chlorella vulgaris microalgae strain, where the obtained results demonstrate its outperformance compared with other methods. More specifically, the average error in the estimation obtained by the proposed approach is 1.54, whereas those obtained by the Gaussian process and gray-scale-based methods are 2.16 and 3.68, respectively.

HEAVY METAL POLLUTANT SORPTION IN AQUATIC ENVIRONMENT BY MICROALGAE CONSORTIUM

Industrial waste that contains heavy metal can cause environmental problem because of its toxicity, persistency and accumulation level in the environment. Biosorption process is highly influenced by temperature, pH, light, contact time, and ratio of surface area. Microalgae which possess two functional groups that are able to react on metal ion in a solution can be exploited to overcome environmental pollution due to heavy metal compound. Closed cultivation system in a photobioreactor is utilized to overcome contamination and evaporation problems on open pond system. Heavy metal analysis is conducted by utilizing Atomic Absorption Spectroscopy (AAS), Fourier Transform Infra-Red (FTIR), and Scanning Electron Microscope (SEM). This article provides information on biosorption as alternative technology to overcome heavy metal in water areas with no side effects on the environment with advantages of the absence of secondary pollutants, high level of efficiency, and relatively economic compared to physic-chemical method heavy metal removal methods

CO2 Utilization Strategy for Sustainable Cultivation of Mushrooms and Lettuces

Mushroom cultivation generates a large amount of CO2 that can be used sustainably. The objective of this study was to use actual cultivation and simulation to find a sustainable cultivation method that uses the CO2 generated by king oyster mushrooms for the production of romaine lettuces. A closed cultivation system consisting of one mushroom chamber, three lettuce chambers, and one gas-mixing chamber was used. Two cultivation conditions, non-continuous and continuous, were analyzed. The non-continuous system cultivated 15 lettuces and 12 mushroom bottles at a time every 25 and 16 days, respectively. The continuous system cultivated three lettuces and mushroom bottles every five and four days, respectively, so that each chamber contained mushrooms or lettuces at each growth stage. The CO2 concentrations in the lettuce and mushroom chambers were stably maintained above 1000 μmol∙mol−1 and below 2000 μmol∙mol−1 in the continuous system. Mathematical models were developed to analyze the CO2 concentration in each chamber. The shoot dry weight of lettuces grown in the mixed cultivation were 48.0%, 21.9%, 19.7%, and 18.1% at 10, 15, 20, and 25 days after transplanting, respectively, higher than those in the lettuce-only cultivation. Compared to mushroom-only cultivation, mixed cultivation reduced the accumulated CO2 emissions into the air by 80.6%. Thus, using CO2 from mushrooms to cultivate lettuce in a continuous cultivation system could reduce CO2 emissions into the air and enable mixed cultivation of mushrooms and lettuces, achieving sustainable agriculture.

Atractylodes lancea volatiles induce physiological responses in neighboring peanut plant during intercropping

Plant volatiles serve as airborne semiochemicals, bridging the interactions between the plant and environment. Intercropping of a Chinese medicinal herb, Atractylodes lancea, with peanut plants greatly improves peanut growth, leading to a reduction of soil-borne disease. The underlying mechanism of peanut responding to the intercropped A. lancea is unknown. We here explored the response of the above- and belowground peanut parts to volatiles produced by the aboveground parts of A. lancea. Closed cultivation system was used. Composition of volatiles released by A. lancea plant was first determined using headspace solid phase microextraction–gas chromatography/mass spectrometry (SPME-GC-MS). Then, physiological responses of peanut were explored via enzymes activity assay and root secretions. Changes in the peanut rhizosphere fungal and bacterial communities were analyzed by Illumina sequencing. The intercropped A. lancea volatiles induced a physiological response in peanut, which includes the increased catalase and phenylalanine ammonia lyase activity in peanut leaf, and improvement of peanut growth. Secretion of organic acids by the peanut root was increased in response to volatile treatment. Pyrosequencing of the whole internal transcribed spacer and 16S rRNA amplicons revealed significant differences in microbial diversity and composition in peanut rhizosphere upon volatile treatment. In the intercropping, A. lancea volatiles play a key role in influencing the growth of a neighbouring peanut plant, e.g., increasing biomass and affecting root colonization by soil microorganisms, which may increase plant protection against pathogens. Intercropping patterns could be designed accordingly to increase crop performance.

Short-term Low Oxygen Treatment Immediately After Planting Alters Asparagus officinalis var. UC157’s Harvest Timing

To control asparagus harvest timing, we investigated the effects of short-term low (5%) oxygen (O2) treatment in the cultivation area on asparagus growth and yield using a closed cultivation system. During 120 days of cultivation, low O2 treatments were initiated at 0 to 4, 20 to 24, and 40 to 44 days after planting (DAP). The sprouting spears and control crown yield gradually decreased with increasing DAP. However, low O2 treatment at 0 to 4 DAP significantly delayed the decrease until 80 DAP, although the total yield did not change during cultivation. In contrast, low O2 treatments at 20 to 24 and 40 to 44 DAP did not affect yield performance. Taken together, short-term low O2 treatment immediately after planting can change the harvest timing of white asparagus and can be used for effective asparagus culturing in a closed system, such as a plant factory.

High density long-term cultivation of Chlorella vulgaris SAG 211-12 in a novel microgravity-capable membrane raceway photobioreactor for future bioregenerative life support in SPACE

Hybrid life support systems are of great interest for future far-distant space exploration missions to planetary surfaces, e.g. Mars, planned until 2050. By synergistically combining physicochemical and biotechnological algae-based subsystems, an essential step towards the closure of the carbon loop in environmental control and life support systems (ECLSS) shall be accomplished, offering a wide beneficial potential for ECLSS through the utilization of oxygenic photosynthesis: O2 and potential human food can be formed in-situ from CO2 and water. The wild type green alga Chlorella vulgaris strain SAG 211-12 was selected as model microorganism due to its photoautotrophic growth, high biomass yield, cultivation flexibility and long-term cultivation robustness. The current study presents for the first time a stable xenic long-term processing of microalgae in a novel microgravity capable membrane raceway photobioreactor for 188 days with the focus on algal growth kinetics and gas evolution. In particular, culture homogeneity and viability were monitored and evaluated during the whole cultivation process due to their putative crucial impact on long-term functionality and efficiency of a closed cultivation system. Based on a specially designed cyclic batch cultivation process for SAG 211-12, a successive biomass growth up to a maximum of 12.2 g l−1 with a max. global volumetric productivity of 1.3 g l−1 d−1 was reached within the closed loop system. The photosynthetic capacity was assessed to a global molar photosynthetic quotient of 0.31. Furthermore, cultivation parameters for a change from batch to continuous processing at high biomass densities and proliferation rates are introduced. The presented µgPBR miniature plant and the developed high throughput cultivation process are planned to be tested under real space conditions within the PBR@LSR project (microgravity and cosmic radiation) aboard the International Space Station with an operation period of up to 180 days to investigate the impact on long-term system stability.

Efficient GMP compliant expansion of mesenchymal stromal cells (MSCs) from umbilical cord, bone marrow and adipose tissue using a closed cultivation system.

Background & Aim Human mesenchymal stem cells (MSCs) hold great promise for clinical use and cell therapy applications and can be isolated from multiple tissue, e.g. bone marrow (BM), umbilical cord (UC) or adipose tissue (AT). As there is a high number of clinical trials and applications using MSCs, quality and safety of the resulting cellular products are indispensable. The xeno-free MSC-Brew GMP Medium following the recommendations of USP on ancillary materials enables a standardized expansion and culture of MSC from different tissue sources. To increase the level of process standardization and product safety we developed the CliniMACS Prodigy® Adherent Cell Culture System for GMP compliant isolation and cultivation of adherent cells using a closed single used tubing set. The process includes the flexible combination of the following modules: - Density Gradient Centrifugation (DGC) - Surface Coating - Inoculation - Culture - Media Change - Harvest Here we show that MSCs from different tissue sources (AT, BM, UC) can be expanded and passaged using the CliniMACS Prodigy® Adherent Cell Culture System from primary tissue or single-cell suspensions combining the process modules in a flexible way. Methods, Results & Conclusion BM-MSCs were isolated via DGC and subsequent adherence to plastic using semi-automatic feeding, harvesting and reseeding procedures. AT-MSCs as well as UC-MSCs were isolated and expanded from single-cell suspensions obtained after enzymatic digestion. UC was digested using an optimized and automated procedure using the gentle MACSTM Dissociator. Subsequently, MSCs were isolated from single cell suspension using the flexible cultivation procedure of the CliniMACS Prodigy® Adherent Cell Culture System. Resulting cells displayed an MSC specific phenotype as defined by the ISCT consortium. Furthermore MSCs revealed their immunomodulatory potential as assessed by in vitro assay and multi-color flow cytometric analysis. With the CliniMACS Prodigy®, MSCs can be expanded in large scales, thus providing high numbers of cells for consecutive clinical applications.

97 - GMP compliant stable and efficient expansion of mesenchymal stromal cells (MSC) in a closed cultivation system

Human mesenchymal stem cells (MSCs) hold great promise for clinical use and cell therapy applications. To ensure highest quality and safety of the resulting cellular products, MSCs have to be maintained under standardized cultivation conditions and procedures. To this, we have developed our xeno-free MSC-Brew GMP medium following the recommendations of USP <1043> on ancillary materials, thus enabling isolation and expansion of MSCs from various tissue sources (e.g. human bone marrow, adipose tissue and umbilical cord) for clinical research use.

Exposure of kale root to NaCl and Na2SeO3 increases isothiocyanate levels and Nrf2 signalling without reducing plant root growth

A plant factory is a closed cultivation system that provides a consistent and modified environment for plant growth. We speculated that treatment of kale (Brassica oleracea) grown in a plant factory with NaCl, Na2SeO3, or both would increase the bioactive phytochemical levels including glucosinolates (GLSs) and isothiocyanates (ITCs), the key molecules in cancer prevention. The kale was harvested and analysed after treatment with NaCl and Na2SeO3 alone or in combination for 1 or 2 weeks. Exposure to NaCl alone but not Na2SeO3 increased plant root growth. Levels of sinigrin were increased by a 2-week exposure to Na2SeO3 alone or in combination with NaCl, whereas no changes were observed in glucoraphanin and gluconasturtiin gluconasturtiin levels. Importantly, the ITC concentration was affected by 2-week treatment with both compounds. To evaluate the bioactivity of kale, HepG2 human hepatoma cells were treated with plant extract for 6 h. Only the extract of kale roots exposed to a combination NaCl and Na2SeO3 for 2 weeks showed an increased expression of nuclear factor erythroid 2-related factor (Nrf2), which regulates genes encoding antioxidant proteins. These data suggest that co-treatment with NaCl and Na2SeO3 increased the ITC content and chemopreventive effects of kale root.

231 - GMP compliant stable and efficient expansion of mesenchymal stromal cells (MSC) in a closed cultivation system

231 - GMP compliant stable and efficient expansion of mesenchymal stromal cells (MSC) in a closed cultivation system

Comparison of Two Harvesting Methods for the Continuous Production of Indigo Plant (Polygonum Tinctorium) Leaves in a Closed-Cultivation System

Although the blue dye indigo has been chemically synthesized for over a century, there is an increasing interest in the indigo plant (Polygonum tinctorium) as a source of natural dyes and medicines. To maintain a stable supply of P. tinctorium throughout the year, we examined the effect of two harvesting methods on the leaf yield of this plant under a closed-cultivation system. With method 8c-M, all shoot branches &gt;8 cm of the stem bottom were harvested and under method 2b-M, all branches, but two, were harvested at the stem bottom. Both methods enabled sustainable leaf yields from the same plants over 1 year. The total weight of shoot branches harvested by 8c-M was 1.86–3.11 times higher that of shoot branches harvested by 2b-M. Harvesting by 8c-M resulted in shoot branch weights lower than those from plants harvested with the 2b-M. Leaf/shoot ratio was increased in plants harvested by 8c-M. The content of indican, the precursor of indigo, in leaves was not significantly different between the two harvesting methods. Our data may provide a new continuous cultivation method of leaf crops all over years in controlled-cultivation systems.

Mushroom Biomass: Some Clinical Implications of β-Glucans and Enzymes

Mushrooms have attracted market attention because they are a potential source of bioactive compounds able to perform several functions in organisms with benefits for the health of the consumer. Cultivation processes vary according a) industrial fermentation - in large vats to produce extracted form of mushrooms or b) closed cultivation system - individually grown in jars on an aseptic “substrate” with controlled lighting and irrigation to produce a biomass form of mushrooms. Biomass is the mycelium with primordia (young fruiting body - before the mushroom blooms) containing all the nutrients and active compounds, including β-glucans, enzymes and secondary metabolites. The classification of mushroom biomass varies according to the presentation; the biomass can be classified as a “food” if in powder form or, classified as a “dietary supplement” in tablet form. While tablet mushroom biomass is considered a dietary supplement, mushroom extracts are designated pharmaceutical compounds, pharmanutrients or nutraceuticals. Here we illustrate the difference between mushrooms in the biomass and extract forms, the similarities and differences on its content on enzymes, secondary metabolites and on β-glucans, as a soluble and fermentable fibre. Of particular note is the rich enzyme activity in the biomass form of mushrooms. Such activity includes enzymes that prevent oxidative stress (superoxide dismutase), enzymes that prevent cellular growth (protease, glucoamylase) and enzymes that promote detoxification (cytochrome P-450, peroxidase, glucose-2-oxidase). β-glucans have been proposed to act as “biological response modifiers” based on their effects on the immune system, and its role in the prevention and treatment of various metabolic syndrome-linked diseases. This review focuses also on some described health-promoting potential of mushroom biomass, all through immunomodulation. The role of intestinal microbiota is enhanced.

Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants.

The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements. Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.

ChemInform Abstract: A Perspective on Gaseous Biofuel Production from Micro‐Algae Generated from CO2 from a Coal‐Fired Power Plant

AbstractReview: 48 refs.