Short-term adaptation as a tool to improve bioethanol production using grass press-juice as fermentation medium.

Grass raw materials collected from grasslands cover more than 30% of Europe's agricultural area. They are considered very attractive for the production of different biochemicals and biofuels due to their high availability and renewability. In this study, a perennial ryegrass (Lolium perenne) was exploited for second-generation bioethanol production. Grass press-cake and grass press-juice were separated using mechanical pretreatment, and the obtained juice was used as a fermentation medium. In this work, Saccharomyces cerevisiae was utilized for bioethanol production using the grass press-juice as the sole fermentation medium. The yeast was able to release about 11 g/L of ethanol in 72 h, with a total production yield of 0.38 ± 0.2 gEthanol/gsugars. It was assessed to improve the fermentation ability of Saccharomyces cerevisiae by using the short-term adaptation. For this purpose, the yeast was initially propagated in increasing the concentration of press-juice. Then, the yeast cells were re-cultivated in 100%(v/v) fresh juice to verify if it had improved the fermentation efficiency. The fructose conversion increased from 79 to 90%, and the ethanol titers reached 18 g/L resulting in a final yield of 0.50 ± 0.06 gEthanol/gsugars with a volumetric productivity of 0.44 ± 0.00 g/Lh. The overall results proved that short-term adaptation was successfully used to improve bioethanol production with S. cerevisiae using grass press-juice as fermentation medium. KEY POINTS: • Mechanical pretreatment of grass raw materials • Production of bioethanol using grass press-juice as fermentation medium • Short-term adaptation as a tool to improve the bioethanol production.

Simultaneous fermentation and enzymatic biocatalysis-a useful process option?

Biotransformation with enzymes and de novo syntheses with whole-cell biocatalysts each have specific advantages. These can be combined to achieve processes with optimal performance. A recent approach is to perform bioconversion processes and enzymatic catalysis simultaneously in one-pot. This is a well-established process in the biorefinery, where starchy or cellulosic material is degraded enzymatically and simultaneously used as substrate for microbial cultivations. This procedure leads to a number of advantages like saving in time but also in the needed equipment (e.g., reaction vessels). In addition, the inhibition or side-reaction of high sugar concentrations can be overcome by combining the processes. These benefits of coupling microbial conversion and enzymatic biotransformation can also be transferred to other processes for example in the sector of biofuel production or in the food industry. However, finding a compromise between the different requirements of the two processes is challenging in some cases. This article summarises the latest developments and process variations.

Optimizing Luminous Transmittance of a Three-Dimensional-Printed Fixed Bed Photobioreactor.

The development of innovative production processes and the optimization of photobioreactors play an important role in generating industrial competitive production technologies for phototrophic biofilms. With emerse photobioreactors a technology was introduced that allowed efficient surface attached cultivation of terrestrial cyanobacteria. However, the productivity of emerse photobioreactors depends on the available cultivation surface. By the implementation of biocarriers to the bioreactor volume, the cultivation surface can be increased which potentially improves productivity and thus the production of valuable compounds. To investigate the surface attached cultivation on biocarriers new photobioreactors need to be developed. Additive manufacturing (AM) offers new opportunities for the design of photobioreactors but producing the needed transparent parts can be challenging using AM techniques. In this study an emerse fixed bed photobioreactor was designed for the use of biocarriers and manufactured using different AM processes. To validate the suitability of the photobioreactor for phototrophic cultivation, the optical properties of three-dimensional (3D)-printed transparent parts and postprocessing techniques to improve luminous transmittance of the components were investigated. We found that stereolithography 3D printing can produce parts with a high luminous transmittance of over 85% and that optimal postprocessing by sanding and clear coating improved the clarity and transmittance to more than 90%. Using the design freedom of AM resulted in a bioreactor with reduced part count and improved handling. In summary, we found that modern 3D-printing technologies and materials are suitable for the manufacturing of functional photobioreactor prototypes.

Towards bioprocess engineering of cable bacteria: Establishment of a synthetic sediment.

Cable bacteria, characterized by their multicellular filamentous growth, are prevalent in both freshwater and marine sediments. They possess the unique ability to transport electrons over distances of centimeters. Coupled with their capacity to fix CO2 and their record-breaking conductivity for biological materials, these bacteria present promising prospects for bioprocess engineering, including potential electrochemical applications. However, the cultivation of cable bacteria has been limited to their natural sediment, constraining their utility in production processes. To address this, our study designs synthetic sediment, drawing on ion exchange chromatography data from natural sediments and existing literature on the requirements of cable bacteria. We examined the effects of varying bentonite concentrations on water retention and the impacts of different sands. For the first time, we cultivated cable bacteria on synthetic sediment, specifically the freshwater strain Electronema aureum GS. This cultivation was conducted over 10 weeks in a specially developed sediment bioreactor, resulting in an increased density of cable bacteria in the sediment and growth up to a depth of 5 cm. The creation of this synthetic sediment paves the way for the reproducible cultivation of cable bacteria. It also opens up possibilities for future process scale-up using readily available components. This advancement holds significant implications for the broader field of bioprocess engineering.

New insights into the influence of pre-culture on robust solvent production of C. acetobutylicum.

Clostridia are known for their solvent production, especially the production of butanol. Concerning the projected depletion of fossil fuels, this is of great interest. The cultivation of clostridia is known to be challenging, and it is difficult to achieve reproducible results and robust processes. However, existing publications usually concentrate on the cultivation conditions of the main culture. In this paper, the influence of cryo-conservation and pre-culture on growth and solvent production in the resulting main cultivation are examined. A protocol was developed that leads to reproducible cultivations of Clostridium acetobutylicum. Detailed investigation of the cell conservation in cryo-cultures ensured reliable cell growth in the pre-culture. Moreover, a reason for the acid crash in the main culture was found, based on the cultivation conditions of the pre-culture. The critical parameter to avoid the acid crash and accomplish the shift to the solventogenesis of clostridia is the metabolic phase in which the cells of the pre-culture were at the time of inoculation of the main culture; this depends on the cultivation time of the pre-culture. Using cells from the exponential growth phase to inoculate the main culture leads to an acid crash. To achieve the solventogenic phase with butanol production, the inoculum should consist of older cells which are in the stationary growth phase. Considering these parameters, which affect the entire cultivation process, reproducible results and reliable solvent production are ensured. KEY POINTS: • Both cryo- and pre-culture strongly impact the cultivation of C. acetobutylicum • Cultivation conditions of the pre-culture are a reason for the acid crash • Inoculum from cells in stationary growth phase ensures shift to solventogenesis.

Beneficial applications of biofilms.

Many microorganisms live in the form of a biofilm. Although they are feared in the medical sector, biofilms that are composed of non-pathogenic organisms can be highly beneficial in many applications, including the production of bulk and fine chemicals. Biofilm systems are natural retentostats in which the biocatalysts can adapt and optimize their metabolism to different conditions over time. The adherent nature of biofilms allows them to be used in continuous systems in which the hydraulic retention time is much shorter than the doubling time of the biocatalysts. Moreover, the resilience of organisms growing in biofilms, together with the potential of uncoupling growth from catalytic activity, offers a wide range of opportunities. The ability to work with continuous systems using a potentially self-advancing whole-cell biocatalyst is attracting interest from a range of disciplines, from applied microbiology to materials science and from bioengineering to process engineering. The field of beneficial biofilms is rapidly evolving, with an increasing number of applications being explored, and the surge in demand for sustainable and biobased solutions and processes is accelerating advances in the field. This Review provides an overview of the research topics, challenges, applications and future directions in beneficial and applied biofilm research.

Autotrophic Production of the Sesquiterpene α-Humulene with Cupriavidus necator in a Controlled Bioreactor.

Cupriavidus necator is a facultative chemolithotrophic organism that grows under both heterotrophic and autotrophic conditions. It is becoming increasingly important due to its ability to convert CO2 into industrially valuable chemicals. To translate the potential of C. necator into technical applications, it is necessary to optimize and scale up production processes. A previous proof-of-principle study showed that C. necator can be used for the de novo production of the terpene α-humulene from CO2 up to concentrations of 11 mg L-1 in septum flasks. However, an increase in final product titer and space-time yield will be necessary to establish an economically viable industrial process. To ensure optimized growth and production conditions, the application of an improved process design in a gas bioreactor with the control of pH, dissolved oxygen and temperature including a controlled gas supply was investigated. In the controlled gas bioreactor, the concentration of α-humulene was improved by a factor of 6.6 and the space-time yield was improved by a factor of 13.2. These results represent an important step toward the autotrophic production of high-value chemicals from CO2. In addition, the in situ product removal of α-humulene was investigated and important indications of the critical logP value were obtained, which was in the range of 3.0-4.2.

A spectroscopic response factor-based toluidine blue assay towards a universal assay protocol for sulfated polysaccharides: Application to fucoidan content in crude extract.

Sulfated polysaccharides (SPS) have attracted a lot of interest because of their diverse pharmacological functions. Numerous scientific studies have shown that SPS exhibit better biological activity than those that are not sulfated, such as immunomodulatory, anti-viral, and antioxidant activities. A crucial step to a better understanding of the mechanism of action and health effects is the production of high purity SPS. This calls for the development of selective assay techniques that can identify SPS preferentially without being influenced by other substances or the co-extracted polysaccharides. A universal modified toluidine blue (TB) assay was developed in this study to detect SPS. The assay procedures were conducted using different SPS standards including fucoidans from different biogenic sources, in addition to heparin and dextran sulfate. Spectroscopic response factor was calculated for each SPS which showed very good correlation (R2 = 0.998) with the corresponding sulfation degree. The proposed method was applied for determination of SPS content of crude fucoidan product using five different SPS standards. The method was cross validated by conducting ANOVA test to the obtained % recovery revealing that there is no significant difference between the results obtained by identical reference standard and four nonidentical natural SPS standards. This is the first report of a selective universal assay of SPS that enables the selective determination of SPS using a nonidentical reference standard.

A new easy method for determination of surface adhesion of phototrophic biofilms.

Terrestrial cyanobacteria grow as phototrophic biofilms and offer a wide spectrum of interesting products. For cultivation of phototrophic biofilms different reactor concepts have been developed in the last years. One of the main influencing factors is the surface material and the adhesion strength of the chosen production strain. In this work a flow chamber was developed, in which, in combination with optical coherence tomography and computational fluid dynamics simulation, an easy analysis of adhesion forces between different biofilms and varied surface materials is possible. Hereby, differences between two cyanobacteria strains and two surface materials were shown. With longer cultivation time of biofilms adhesion increased in all experiments. Additionally, the content of extracellular polymeric substances was analyzed and its role in surface adhesion was evaluated. To test the comparability of obtained results from the flow chamber with other methods, analogous experiments were conducted with a rotational rheometer, which proved to be successful. Thus, with the presented flow chamber an easy to implement method for analysis of biofilm adhesion was developed, which can be used in future research for determination of suitable combinations of microorganisms with cultivation surfaces on lab scale in advance of larger processes.

Metabolism of natural and synthetic bioactive compounds in Cunninghamella fungi and their applications in drug discovery.

Investigation of xenobiotic metabolism is a key step for drug discovery. Since the in vivo investigations may be associated with harmful effects attributed to production of toxic metabolites, it is deemed necessary to predict their structure especially at the preliminary clinical studies. Furthermore, the application of microorganisms that are capable of metabolizing drugs mimic human metabolism and consequently may predict possible metabolites. The genus Cunninghamella has been proven to be a potential candidate, which mimics xenobiotic metabolism occurring inside the human body, including phase I and II metabolic reactions. Moreover, biotransformation with Cunninghamella showed chemical diversity, where a lot of products were detected in relation to the initial substrates after being modified by oxidation, hydroxylation, and conjugation reactions. Some of these products are more bioactive than the parent compounds. The current review presents a comprehensive literature overview regarding the Cunninghamella organisms as biocatalysts, which simulate mammalian metabolism of natural secondary and synthetic compounds.

Methyl jasmonate elicitation effect on the metabolic profile of cambial meristematic cells culture derived from sweet basil (Ocimum basilicum L.) in relation to antioxidant activity: Untargeted metabolomics study in a time-based approach.

The undifferentiated cambial meristematic cell (CMC) has been recognized as a value-added production platform for plant natural products in comparison to the dedifferentiated plant cell line (DDC). In a time-based approach at 0, 24, 48, and 72 h, the present study aimed at investigating the phytochemical metabolome of methyl jasmonate (MeJA)-elicited CMC cultures derived from sweet basil (Ocimum basilicum L.), including primary and secondary metabolites analyzed using GC/TOF-MS post-silylation and RP-UPLC-C18-FT-MS/MS, respectively, as well as the analysis of aroma composition using headspace SPME-GC-MS. The results revealed a stress response in primary metabolism manifested by an increase in amino and organic acids reaching their maximum levels after 48 (1.3-fold) and 72 (1.7-fold) h, respectively. In addition, phenolic acids (e.g., sagerinic acid, rosmarinic acid, and 3-O-methylrosmarinic acid) followed by flavonoid aglycones (e.g., salvigenin and 5,6,4'-trihydroxy-7,3'-dimethoxyflavone) were the most abundant with prominent increases at 48 (1.2-fold) and 72 (2.1-fold) h, respectively. The aroma was intensified by the elicitation along the time, especially after 48 and 72 h. Furthermore, multivariate data analyses, including principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) confirmed elicitation effect, especially post 48 and 72 h. The study further assessed the effect of MeJA elicitation on the antioxidant and polyphenolic content. The cultures at 48 h demonstrated a significant (p < 0.05) antioxidant activity concurrently with correlation with total polyphenolic content using Pearson's correlation. Our study provides new insights to the elicitation impact on primary and secondary metabolism, in addition to aroma profile, to orchestrate the stress response and in relation to antioxidant effect.

Fucoidan mitigates gastric ulcer injury through managing inflammation, oxidative stress, and NLRP3-mediated pyroptosis.

This study aimed to elucidate the gastro-protective effect of fucoidan against ethanol-induced gastric ulcer mediated via NLRP3-induced pyroptosis as an underlying mechanism, not yet assessed in prior research. Forty-eight male Albino mice were divided into six groups: Group I (normal control), group II (Ulcer/ethanol control), group III (Omeprazole + ethanol), group IV (fucoidan 25 mg + ethanol), group V (fucoidan 50 mg + ethanol) and group VI (fucoidan only). Fucoidan was administered orally for seven consecutive days followed by ulcer induction by a single oral dose of ethanol. Using colorimetric analysis, ELISA, qRT-PCR, histological assessment, and immunohistochemical studies, the results revealed that ethanol-induced ulcer exhibited an ulcer score of 42.5 ± 5.1 and a significant increase (p < 0.05) in malondialdehyde (MDA), nuclear factor kappa B (NF-κB), and interleukin 6 (IL-6) with a significant decrease in the gastro-protective mediators, prostaglandin E2 (PGE2), superoxide dismutase (SOD) and glutathione (GSH), accompanied with an increase in NLRP3, interleukin 1ß (IL-1ß), interleukin 18 (IL-18), caspase 1, caspase 11, gasdermin D, and toll-like receptor 4 (TLR4), compared with the normal control. Pre-treatment with fucoidan showed a comparable result with omeprazole. Additionally, pre-treatments elevated the levels of the gastro-protective mediators and lessened oxidative stress, relative to the positive control findings. Conclusively, fucoidan has a promising gastro-protective role by inhibiting inflammation and pyroptosis.

Fucoidan in Pharmaceutical Formulations: A Comprehensive Review for Smart Drug Delivery Systems.

Fucoidan is a heterogeneous group of polysaccharides isolated from marine organisms, including brown algae and marine invertebrates. The physicochemical characteristics and potential bioactivities of fucoidan have attracted substantial interest in pharmaceutical industries in the past few decades. These polysaccharides are characterized by possessing sulfate ester groups that impart negatively charged surfaces, low/high molecular weight, and water solubility. In addition, various promising bioactivities have been reported, such as antitumor, immunomodulatory, and antiviral effects. Hence, the formulation of fucoidan has been investigated in the past few years in diverse pharmaceutical dosage forms to be able to reach their site of action effectively. Moreover, they can act as carriers for various drugs in value-added drug delivery systems. The current work highlights the attractive biopharmaceutical properties of fucoidan being formulated in oral, inhalable, topical, injectable, and other advanced formulations treating life-quality-affecting diseases. Therefore, the present work points out the current status of fucoidan pharmaceutical formulations for future research transferring their application from in vitro and in vivo studies to clinical application and market availability.

Characterization and Cytotoxic Activity of Microwave-Assisted Extracted Crude Fucoidans from Different Brown Seaweeds.

Microwave-assisted extraction (MAE) is recognized as a green method for extraction of natural products. The current research aimed to explore the MAE for fucoidans extraction from different brown seaweeds, including Fucus vesiculosus, F. spiralis, and Laminaria saccharina. Following several solvent-extraction pre-treatment steps and MAE optimization, the algal biomasses were extracted in a ratio of 1:25 in 0.1 M HCl containing 2 M CaCl2 for 1.0 min. The results showed that L. saccharina's extract was different from the others, regarding the highest sugar content reached 0.47 mg glucose equivalent/mg extract being confirmed by monosaccharide composition analysis and the lowest fucoidan content and sulfation degree at 0.09 mg/mg extract and 0.13, respectively. Moreover, these findings were confirmed by tentative structural elucidation based on Fourier-transform infrared spectrometry which also showed a different spectrum. However, the MAE enhanced melanoidins formation in products, which was confirmed by the intense band at 1420 cm-1. Interestingly, the results of monomeric composition showed that fucoidan extract by MAE from F. vesiculosus belonged to sulfated galactofucans which are known for their potential bioactivities. Furthermore, the cytotoxic activity of the four fucoidans in concentrations ranging from 4.9 µg/mL to 2500 µg/mL was investigated and correlated with the chemical characterization showing that F. vesiculosus_MAE fucoidan was the most potent and safest. The current research revealed the chemical heterogeneity of fucoidans regarding taxonomical class and used greener extraction method of fucoidans toward the achievement of the UN sustainability goals.

Process Technologies of Cyanobacteria.

Although the handling and exploitation of cyanobacteria is associated with some challenges, these phototrophic bacteria offer great opportunities for innovative biotechnological processes. This chapter covers versatile aspects of working with cyanobacteria, starting with up-to-date in silico and in vitro screening methods for bioactive substances. Subsequently, common conservation techniques and vitality/viability estimation methods are compared and supplemented by own data regarding the non-invasive vitality evaluation via pulse amplitude modulated fluorometry. Moreover, novel findings about the influence the state of the pre-cultures have on main cultures are presented. The following sub-chapters deal with different photobioreactor-designs, with special regard to biofilm photobioreactors, as well as with heterotrophic and mixotrophic cultivation modes. The latter topic provides information from literature on successfully enhanced cyanobacterial production processes, augmented by own data.

Fucoidan's Molecular Targets: A Comprehensive Review of Its Unique and Multiple Targets Accounting for Promising Bioactivities Supported by In Silico Studies.

Fucoidan is a class of multifunctional polysaccharides derived from marine organisms. Its unique and diversified physicochemical and chemical properties have qualified them for potential and promising pharmacological uses in human diseases, including inflammation, tumors, immunity disorders, kidney diseases, and diabetes. Physicochemical and chemical properties are the main contributors to these bioactivities. The previous literature has attributed such activities to its ability to target key enzymes and receptors involved in potential disease pathways, either directly or indirectly, where the anionic sulfate ester groups are mainly involved in these interactions. These findings also confirm the advantageous pharmacological uses of sulfated versus non-sulfated polysaccharides. The current review shall highlight the molecular targets of fucoidans, especially enzymes, and the subsequent responses via either the upregulation or downregulation of mediators' expression in various tissue abnormalities. In addition, in silico studies will be applied to support the previous findings and show the significant contributors. The current review may help in understanding the molecular mechanisms of fucoidan. Also, the findings of this review may be utilized in the design of specific oligomers inspired by fucoidan with the purpose of treating life-threatening human diseases effectively.

Municipal green waste as substrate for the microbial production of platform chemicals.

In Germany alone, more than 5·106 tons of municipal green waste is produced each year. So far, this material is not used in an economically worthwhile way. In this work, grass clippings and tree pruning as examples of municipal green waste were utilized as feedstock for the microbial production of platform chemicals. A pretreatment procedure depending on the moisture and lignin content of the biomass was developed. The suitability of grass press juice and enzymatic hydrolysate of lignocellulosic biomass pretreated with an organosolv process as fermentation medium or medium supplement for the cultivation of Saccharomyces cerevisiae, Lactobacillus delbrueckii subsp. lactis, Ustilago maydis, and Clostridium acetobutylicum was demonstrated. Product concentrations of 9.4 gethanol L-1, 16.9 glactic acid L-1, 20.0 gitaconic acid L-1, and 15.5 gsolvents L-1 were achieved in the different processes. Yields were in the same range as or higher than those of reference processes grown in established standard media. By reducing the waste arising in cities and using municipal green waste as feedstock to produce platform chemicals, this work contributes to the UN sustainability goals and supports the transition toward a circular bioeconomy.

Fermentative α-Humulene Production from Homogenized Grass Clippings as a Growth Medium.

Green waste, e.g., grass clippings, is currently insufficiently recycled and has untapped potential as a valuable resource. Our aim was to use juice from grass clippings as a growth medium for microorganisms. Herein, we demonstrate the production of the sesquiterpene α-humulene with the versatile organism Cupriavidus necator pKR-hum on a growth medium from grass clippings. The medium was compared with established media in terms of microbial growth and terpene production. C. necator pKR-hum shows a maximum growth rate of 0.43 h-1 in the grass medium and 0.50 h-1 in a lysogeny broth (LB) medium. With the grass medium, 2 mg/L of α-humulene were produced compared to 10 mg/L with the LB medium. By concentrating the grass medium and using a controlled bioreactor in combination with an optimized in situ product removal, comparable product concentrations could likely be achieved. To the best of our knowledge, this is the first time that juice from grass clippings has been used as a growth medium without any further additives for microbial product synthesis. This use of green waste as a material represents a new bioeconomic utilization option of waste materials and could contribute to improving the economics of grass biorefineries.

Insight into Fucoidan-Based PEGylated PLGA Nanoparticles Encapsulating Methyl Anthranilic Acid: In Vitro Evaluation and In Vivo Anti-Inflammatory Study.

A potential fucoidan-based PEGylated PLGA nanoparticles (NPs) offering a proper delivery of N-methyl anthranilic acid (MA, a model of hydrophobic anti-inflammatory drug) have been developed via the formation of fucoidan aqueous coating surrounding PEGylated PLGA NPs. The optimum formulation (FuP2) composed of fucoidan:m-PEG-PLGA (1:0.5 w/w) with particle size (365 ± 20.76 nm), zeta potential (-22.30 ± 2.56 mV), % entrapment efficiency (85.45 ± 7.41), drug loading (51.36 ± 4.75 µg/mg of NPs), % initial burst (47.91 ± 5.89), and % cumulative release (102.79 ± 6.89) has been further investigated for the anti-inflammatory in vivo study. This effect of FuP2 was assessed in rats' carrageenan-induced acute inflammation model. The average weight of the paw edema was significantly lowered (p ≤ 0.05) by treatment with FuP2. Moreover, cyclooxygenase-2 and tumor necrosis factor-alpha immunostaining were decreased in FuP2 treated group compared to the other groups. The levels of prostaglandin E2, nitric oxide, and malondialdehyde were significantly reduced (p ≤ 0.05) in the FuP2-treated group. A significant reduction (p ≤ 0.05) in the expression of interleukins (IL-1ß and IL-6) with an improvement of the histological findings of the paw tissues was observed in the FuP2-treated group. Thus, fucoidan-based PEGylated PLGA-MA NPs are a promising anti-inflammatory delivery system that can be applied for other similar drugs potentiating their pharmacological and pharmacokinetic properties.

Portuino-A Novel Portable Low-Cost Arduino-Based Photo- and Fluorimeter.

A novel portable low-cost Arduino-controlled photo- and fluorimeter for on-site measurements has been developed. The device uses LEDs as a light source and a phototransistor as a light sensor. The circuit is based on the discharge of a capacitor with the photocurrent from the phototransistor. Validation experiments for absorbance measurements were performed by measuring protein concentration using the Bradford method and measuring phosphate ions in water using a commercial test kit. The emission light of the excited fluorescent dyes rhodamine 6G and calcofluor white was measured to validate the usability of the device as a fluorescence photometer. In all validation experiments, similar correlation coefficients and limit of detection could be achieved with the portable photo- and fluorimeter and a laboratory spectrometer and fluorimeter. Real sample analysis was performed, measuring phosphate concentration in freshwater and concentration of green fluorescent protein, extracted from Escherichia coli.