Photo-controlled release hydrogel provides a new strategy for treating tumours. Under the stimulation of external light sources, the ability to release the entrapped drug on time and space on demand has outstanding advantages in improving drug utilisation, optimising treatment, and reducing toxicity and side effects. In this study, a photo-controlled drug delivery system for disulphide cross-linked polyaspartic acid (PASP-SS) hydrogels encapsulating proteinase K (ProK) adsorbed with platinum nanoparticles (PtNPs) was designed. The injectable cysteamine-modified polyaspartic acid (PASP-SH) sol and PtNPs adsorbed by ProK (ProK-PtNPs) as regulatory factors were prepared. Then, ProK-PtNPs and lentinan were dissolved in the sol, and the oxidant was added to the matrix to form the gel in situ quickly after injection. Finally, the degradation of PASP-SS hydrogel by ProK and the controllability of drug release under near-infrared (NIR) light irradiation were elucidated. In vitro degradation of hydrogels and drug release experiments showed that the degradation rate of PASP-SS hydrogel significantly increased and the drug release rate increased significantly under near-infrared radiation. The results of cytotoxicity test showed that PASP-SS, ProK-PtNPs, and lentinan all had more than 90% cell survival rate on NIH3T3, and the lentinan released from the carrier obviously inhibited the proliferation of MCF7. PASP hydrogel has the potential to respond to on-demand light control.
Steroids are the most widely marketed products by the pharmaceutical industry after antibiotics. Steroid hydroxylation is one of the most important functionalizations because their derivatives enable a higher biological activity compared to their less polar non-hydroxylated analogs. Bacterial cytochrome P450s constitute promising biocatalysts for steroid hydroxylation due to their high expression level in common workhorses like Escherichia coli. However, they often suffer from wrong or insufficient regio- and/or stereoselectivity, low activity, narrow substrate range as well as insufficient thermostability, which hampers their industrial application. Fortunately, these problems can be generally solved by protein engineering based on directed evolution and rational design. In this work, an overview of recent developments on the engineering of bacterial cytochrome P450s for steroid hydroxylation is presented.
In this work, the potential for using Millettia pinnata stalk for extracting cellulosic natural fibers and its subsequent use in reinforced composites was studied. The extracted fibers were characterized for its composition, mechanical, thermal stability and morphological properties. Compositional analysis showed that the fibers possessed 54% cellulose, 12% hemicellulose, 15% lignin and 11% ash. The tensile strength of the fiber was 310 MPa, which is comparable to cotton and linen. The tensile strength of the M. pinnata fiber-reinforced polypropylene composites was 17.96 MPa which was similar to other natural fiber-based composites. M. pinnata fibers appear promising for a wide range of applications including textiles and other typical composites applications.
This paper describes the preparation and characterization of MgO and ZnO-based catalysts, pure and mixed in different proportions, supported on γ-Al2O3. Their catalytic performance was studied in the transesterification of soybean oil and castor oil with methanol and butanol, attempting to produce biodiesel. XRD (X-ray diffraction), SEM–EDS (scanning electron microscopy–energy dispersive X-ray spectroscopy), CO2-adsorption and N2-adsorption allowed characterizing the prepared catalysts. The characterization results were in all cases consistent with mesoporous solids with high specific surface area. All the catalysts exhibited good results, especially in the transesterification of castor oil using butanol. For this reaction, the reuse was tested, maintaining high FABE (fatty acid butyl esters) yields after four cycles. This good performance can be attributed to the basic properties of the Mg species, and simultaneously, to the amphoteric properties of ZnO, which allow both triglycerides and free fatty acids to be converted into esters. Using these catalysts, it is possible to obtain second-generation biodiesel, employing castor oil, a raw material that does not compete with the food industry. In addition, butanol can be produced from renewable biomass.
Haematococcus pluvialis is the best source of natural astaxanthin, known as the king of antioxidants. H. pluvialis have four cell forms: spore, motile cell, non-motile cell and akinete. Spores and motile cells are susceptible to photoinhibition and would die under photoinduction conditions. Photoinduction using non-motile cells as seeds could result in a higher astaxanthin production than that using akinetes. However, the mechanism of this phenomenon has not been clarified.
Transcriptome was sequenced and annotated to illustrate the mechanism of this phenomenon. All differentially expressed genes involved in astaxanthin biosynthesis were up-regulated. Particularly, chyb gene was up-regulated by 16-fold, improving the conversion of β-carotene into astaxanthin. Pyruvate was the precursor of carotenoids biosynthesis. Pyruvate kinase gene expression level was increased by 2.0-fold at the early stage of akinetes formation. More changes of gene transcription occurred at the early stage of akinetes formation, 52.7% and 51.9% of total DEGs in control group and treatment group, respectively.
Genes transcription network was constructed and the synthesis mechanism of astaxanthin was clarified. The results are expected to further guide the in-depth optimization of the astaxanthin production process in H. pluvialis by improving pyruvate metabolism.
In many countries, agricultural residues are generated in large quantities, and most of these are underutilized and considered waste, especially in developing countries.
In this study, Curvularia affinis was isolated from the leaves of Phaseolus vulgaris L. beans and identified using 18S rRNA sequencing. C. affinis was tested for exo- and endoglucanase production using biomass of bean waste compared with the use of microcrystalline cellulose (MCC) and carboxymethylcellulose (CMC) as its growth substrates. C. affinis was better able to produce exo- and endoglucanase enzymes on bean waste biomass than on MCC and CMC. The highest activities of exo- and endoglucanase were detected with substrate concentrations 2% using MCC or CMC and with 4% using bean waste. The optimum incubation period for enzymes activity was 6 days with MCC or CMC (activity was 5.90 and 2.99 U/g of exoglucanase and endoglucanase, respectively) and 8 days with bean waste where activity was 3.64 U/g and 0.92 U/g of exoglucanase and endoglucanase, respectively. Exo- and endoglucanase production showed the highest activity at pH 5–6. In process wherein surfactant (Tween 80) was used, the exoglucanase activity gradually increased from 5.92 U/g to 6.20 U/g and then decreased to 5.70 U/g at 0.50% compared with that using the MCC substrate. The exoglucanase activity gradually increased from 3.80 U/g at 0.0% to 4.12 U/g at 0.20% and then decreased to 3.01 U/g at 0.50% Tween 80 using bean waste. Pretreated bean biomass also yielded higher enzyme production than the non-pretreated biomass. Alkaline-pretreated biomass showed the highest enzyme production compared with acid-treated residues, followed by the H2O2-treated ones.
The study concluded that C. affinis produce exo- and endoglucanase enzymes using cheap and abundant biomass of beans. Moreover, optimization of enzymes indicated that pretreatment of biomass bean biomass is a good choice process for enhanced enzymes productivity.
Waste generation tends to surge in quantum as the population and living conditions grow. A group of structurally related chemicals of dibenzofurans and dibenzo-p-dioxins including their chlorinated congeners collectively known as dioxins are among the most lethal environmental pollutants formed during different anthropogenic activities. Removal of dioxins from the environment is challenging due to their persistence, recalcitrance to biodegradation, and prevalent nature. Dioxin elimination through the biological approach is considered both economically and environmentally as a better substitute to physicochemical conventional approaches. Bacterial aerobic degradation of these compounds is through two major catabolic routes: lateral and angular dioxygenation pathways. Information on the diversity of bacteria with aerobic dioxin degradation capability has accumulated over the years and efforts have been made to harness this fundamental knowledge to cleanup dioxin-polluted soils. This paper covers the previous decades and recent developments on bacterial diversity and aerobic bacterial transformation, degradation, and bioremediation of dioxins in contaminated systems.
Members of Bacillaceae family are of major interest in medical industry due to vast antimicrobial peptides they produce as therapeutic agents. For decades, synthetic and natural occurring antibiotics have been used to treat infectious diseases, but heavy dependence on these drugs has led to significant drawbacks which propel continuous development of new antibiotics generation. Recent findings have shown several bacteriocins of Bacillaceae as promising alternatives to the conventional drugs to combat the emergence of new drug-resistant pathogens. In this present review, Bacillaceae bacteriocins’ classification such as lantibiotics and thiazole/oxazole-modified microcins as well as their biochemical characterization such as sensitivity to enzymes, temperature, pH and chemicals are described. This article enlightens on the medical application of several Bacillaceae bacteriocins emphasizing those that underwent and on-going preclinical trials. This review also discusses the development of Bacillaceae bacteriocins production, focusing strains selection and fermentation factors such as inocula size, medium (carbon, nitrogen, minerals sources), temperature, pH, agitation and aeration rate, dissolved oxygen tension (DOT), fermentation time, inducers and mode of operation via various statistical methods for their optimization. It also highlights recent advance in the production of bioengineered and recombinant bacteriocins in bioreactors system which are rarely disclosed in literature.
In this study, the biodegradation of N-acetyl-para-aminophenol also known as acetaminophen (APAP, paracetamol) was studied by bacterial strain Bacillus drentensis strain S1 (accession no. KY623719) isolated from sewage sample.
The Bacillus drentensis strain S1 was isolated from the sewage sample using the enrichment culture method. As per our knowledge this is the first Bacillus drentensis strain reported for the degradation of APAP. In this study a 20-L batch reactor was employed for degradation of APAP. The maximum specific growth rate (μmax) was observed at 400 mg/L concentration of APAP. The pilot-scale anaerobic batch reactor of was stable and self-buffered. The degradation in pilot-scale reactor was slow as compared to batch experiments due to fluctuation in pH and exhaustion of nutrients. Design-Expert® software was used for optimization of conditions for APAP degradation; such as temperature (40 °C), pH (7.0), concentration of APAP (300 g/L) and agitation speed (165 rpm). The FTIR and GC–MS were used to identify the degradation metabolites. The intermediates of degradation like 2-isopropyl-5-methylcyclohexanone and phenothiazine were observed, based on these results the metabolic pathway has been predicted.
The optimization, kinetic, batch study and pilot study indicates the potential of Bacillus drentensis strain S1 for degradation of acetaminophen. The experimental design, optimization and statistical analysis were performed by Design Expert® software. The optimal growth condition for Bacillus drentensis strain S1 was found to be at temperature 40 °C, pH 7, acetaminophen at concentration of 300 (mg/L) and agitation speed 165 rpm. The GC–MS and FTIR was used for identification of metabolites produced during acetaminophen degradation and the partial metabolic pathway for degradation of acetaminophen was also proposed .
The simultaneous and sequential dual elicitation effect on plumbagin production in Plumbago indica L. root cultures, revealed that combination of chitosan (150 mg L−1) with ʟ-alanine (5 mM) or methyl-β-cyclodextrin (MCD; 2 mM) significantly increased plumbagin production, but in the different treatment manners. The simultaneous treatment using chitosan + ʟ-alanine on a 14-day-old culture enhanced plumbagin production to 14.62 mg g−1 DW, while the sequential additions of MCD to a 12-day-old culture followed by chitosan after 48 h enhanced production of plumbagin to 14.33 mg g−1 DW. The plumbagin productivity from both treatments were up to 1.3- and 8-fold higher than the chitosan treated (10.93 mg g−1 DW) and untreated root cultures (1.76 mg g−1 DW). Moreover, the present studies provided new information on the effect of simultaneous and sequential elicitation on plumbagin-producing P. indica root cultures using chitosan in combinations with MCD or ʟ-alanine.
Renewable biomass such as cellulose and chitin are the most abundant sustainable sources of energy and materials. However, due to the low degradation efficiency of these recalcitrant substrates by conventional hydrolases, these biomass resources cannot be utilized efficiently. In 2010, the discovery of lytic polysaccharide monooxygenases (LPMOs) led to a major breakthrough. Currently, LPMOs are distributed in 7 families in CAZy database, including AA9–11 and AA13–16, with different species origins, substrate specificity and oxidative regioselectivity. Effective application of LPMOs in the biotransformation of biomass resources needs the elucidation of the molecular basis of their function. Since the discovery of LPMOs, great advances have been made in the study of their substrate specificity and regioselectivity, as well as their structural basis, which will be reviewed below.
Saccharopolyspora erythraea (S. erythraea) is a Gram-positive bacterium widely used for the production of erythromycin, a potent macrolide antibiotic. However, the mechanism behind erythromycin production is poorly understood. In the high erythromycin-producer strain S. erythraea HL3168 E3, the level of copper ions positively correlates with erythromycin production. To explain this correlation, we performed a genome-based comparison between the wild-type strain NRRL23338 and the mutant strain HL3168 E3, and further characterized the identified gene(s) by targeted genome editing, mRNA transcript analysis, and functional analysis.
The response regulator of the two-component system (TCS) encoded by the gene SACE_0101 in S. erythraea showed high similarity with CopR of TCS CopRS in Streptomyces coelicolor, which is involved in the regulation of copper metabolism. The deletion of SACE_0101 was beneficial for erythromycin synthesis most likely by causing changes in the intracellular copper homeostasis, leading to enhanced erythromycin production. In addition, Cu2+ supplementation and gene expression analysis suggested that SACE_0101 may be involved in the regulation of copper homeostasis and erythromycin production.
The mutation of SACE_0101 gene increased the yield of erythromycin, especially upon the addition of copper ions. Therefore, the two-component system gene SACE_0101 plays a crucial role in regulating copper homeostasis and erythromycin synthesis in S. erythraea.
Bikaverin is a fungal red pigment that presents antimicrobial and antitumor activities. Therefore, this substance could be used as an alternative additive in the food and pharmaceutical industries. The aim of this work was to use response surface methodology to optimize the fermentation conditions and maximize the production of bikaverin in shake flasks. The variables investigated were agitation speed (71–289 rpm), temperature (21–35 °C), and substrate (rice) concentration in the culture medium (16.4–83.6 g/L). The agitation speed had a positive effect on red pigment production, while substrate concentration and temperature had the opposite effect. Maximum bikaverin production was predicted to occur using 289 rpm, 24.3 °C, and 16.4 g/L rice concentration. Experimental validation using 289 rpm, 28 °C, and 20 g/L rice concentration was 6.2% higher than predicted by the model. The present investigation was important for defining the best conditions for the production of bikaverin.
The present study evaluated the potential biological activities of rhizospheric fungi isolated from the Achanakmar Biosphere Reserve, India. Fungus, Talaromyces purpureogenus isolate-ABRF2 from the soil of the Achanakmar biosphere was characterized by using morphological, biochemical and molecular techniques. Fungus was screened for the production of secondary metabolites using a specific medium. The metabolites were extracted using a suitable solvent and each fraction was subsequently evaluated for their antioxidant, antimicrobial, antiproliferative and anti-aging properties. The ethanolic extract depicted the highest antioxidant activity with 83%, 79%, 80% and 74% as assessed by ferric reducing power, 2,2-diphenyl 1-picrylhydrazyl, 2,2′-azino-bis3-ethylbenzthiazoline-6-sulfonic and phosphomolybdenum assays, respectively. Similarly, ethanolic extracts depicted marked antimicrobial activity as compared with standard antibiotics and antifungal agents as well as demonstrated significant antiproliferative property against a panel of mammalian cancer cell lines. Furthermore, different fractions of the purified ethanolic extract obtained using adsorption column chromatography were evaluated for antiproliferative property and identification of an active metabolite in the purified fraction using gas chromatography–mass spectroscopy and nuclear magnetic resonance techniques yielded 3-methyl-4-oxo-pentanoic acid. Thus, the present study suggests that the active metabolite 3-methyl-4-oxo-pentanoic acid extracted from Talaromyces purpureogenus isolate-ABRF2 has a potential antiproliferative, anti-aging, and antimicrobial therapeutic properties that will be further evaluated using in vivo studies in future.
Parametric optimization is an effective way in fermentation process to improve product yield and productivity in order to save time, space and financial resources. In this study, Box–Behnken design was applied to optimize the conditions for lactic acid production by immobilized Lactobacillus pentosus ATCC 8041 cell fermentation. Two quadratic models and response surface methodology were performed to illustrate the effect of each parameters and their interactions on the lactic acid yield and glucose consumption rate in immobilized L. pentosus ATCC 8041 cell fermentation. The maximum lactic acid yield was obtained as 0.938 ± 0.003 g/g glucose with a productivity of 2.213 ± 0.008 g/(L × h) under the optimized conditions of 2.0 mm bead diameter, 5.60 pH, 115.3 g/L initial glucose concentration, and 398.2 mg biomass (CDW) in 100 mL hydrogel. The analysis of variance indicated that the quadratic model was significant and could be used to scale up the fermentation process.
The growth of Aspergillus awamori and Aspergillus oryzae in a self-designed, multi-stacked circular tray solid-state bioreactor (SSB), operating in solid-state fermentation (SSF) conditions at a laboratory scale, was studied. The bioreactor was divided into six layers by six circular perforated trays. Wheat bran was used as both a carrier of bound mycelia and nutrient medium for the growth of A. awamori and A. oryzae. The new tray SSB is equipped with instrumentation (an oxygen (O2)/carbon dioxide (CO2) gas analyser and a thermocouple) to continuously monitor O2 consumption and CO2 and heat evolved, which can directly be used to monitor the fungal biomass. The integrated Gompertz model was used to describe the accumulated evolution of CO2.
The results from the models strongly suggest that the evolved and accumulated CO2 can be used to excellently describe fungal growth. Another important parameter that can be determined by the gas balance method is the respiratory quotient (RQ). This is the ratio of the CO2 evolution rate (CER) to the O2 uptake rate (OUR). The use of CER and OUR confirmed that correlated measurements of microbial activity are available, and the determination of RQ may propose an explanation for differences from expected levels. The kinetic behaviour of the fungal culture, using raw CO2, which represents an accumulation term, was integrated with respect to time and fitted to a Gompertz model, a log-like equation. The model can be used to generate parameter values that may be used to verify the experimental data, and also to simulate and optimise the process.
Overall, A. awamori and A. oryzae have their own ability to degrade and utilise the complex compositions contained in the solid substrate, and fermentation conditions may lead to possible comparisons. In addition, multi-stacked circular tray SSB systems demonstrated an excellent system for further investigations of mass transfer and possibly for large-scale operation, though considerable optimisation work remains to be done; for example, the height/diameter ratio and total number of trays should be optimised.
The goal of cell culture process intensification is to increase volumetric productivity, generally by increasing viable cell density (VCD), cell specific productivity or production bioreactor utilization in manufacturing. In our previous study, process intensification in fed-batch production with higher titer or shorter duration was demonstrated by increasing the inoculation seeding density (SD) from ~ 0.6 (Process A) to 3–6 × 106 cells/mL (Process B) in combination with media enrichment. In this study, we further increased SD to 10–20 × 106 cells/mL (Process C) using perfusion N-1 seed cultures, which increased titers already at industrially relevant levels by 100% in 10–14 day bioreactor durations for four different mAb-expressing CHO cell lines. Redesigned basal and feed media were critical for maintaining higher VCD and cell specific productivity during the entire production duration, while medium enrichment, feeding strategies and temperature shift optimization to accommodate high VCDs were also important. The intensified Process C was successfully scaled up in 500-L bioreactors for 3 of the 4 mAbs, and quality attributes were similar to the corresponding Process A or Process B at 1000-L scale. The fed-batch process intensification strategies developed in this study could be applied for manufacturing of other mAbs using CHO and other host cells.
To explore the differences in the intracellular transcriptional mechanism in carbon-derepressed and wild-type Pichia pastoris strains fed with three different carbon sources. RNA in carbon-derepressed (Δmig1Δmig2Δnrg1-Mit1; Mut) and wild-type (WT) P. pastoris fed with three different carbon sources (dextrose, glycerol, and methanol) were sequenced. Differentially expressed genes (DEGs) associated with these carbon sources were obtained and clustered into modules using weighted gene co-expression network analysis (WGCNA). Signaling pathway enrichment analysis was performed using KEGG, and protein to protein interaction (PPI) network was also constructed. A total of 2536 DEGs were obtained from three intersections, and some of them were enriched in carbon sources and involved in carbon metabolism, secondary metabolisms, and amino acid biosynthesis. Two modules, MEgreenyellow (involved in protease, oxidative phosphorylation, endoplasmic reticulum protein processing, folate carbon pool, and glycerol phospholipid metabolism pathways) and MEmidnightblue (involved in protease, endocytosis, steroid biosynthesis, and hippo signaling pathways) were significantly correlated with the strain type. Eight hub genes and two sub-networks were obtained from PPI network. Sub-network A enriched in proteasomes pathway while sub-network B enriched in ribosome pathway. The genes involved in carbon metabolism, secondary metabolic, and amino acid biosynthesis pathways changed significantly under different carbon sources. The changes in proteasome and ribosome activities play roles in carbohydrate metabolism in the methanol-free PAOX1 start-up Mut strain.
This study aims to optimize strong acid hydrolysis-based production of cellulose nanocrystals (CNCs) and cellulose nanofibers (CNFs) from pre-extracted and fully bleached kraft pulp of loblolly pinewood, the most abundant and commercially significant softwood species in southeastern United States. The effect of four parameters, including acid concentration, temperature, duration and pulp particle size, on the yield and properties of CNCs was investigated using the central composite design (CCD) of response surface methodology (RSM) for process optimization. While CNC yield was significantly affected by acid concentration and hydrolysis temperature and was adequately explained by an empirical model, none of the characteristic properties of CNCs, including crystallinity index, surface charge and particle size, displayed any strong correlation to the process parameters within the experimental ranges tested. At different hydrolysis severities, we not only analyzed the waste streams to determine the extent of holocellulose degradation, but also evaluated the properties of leftover partially hydrolyzed pulp, called cellulosic solid residues (CSR), to gauge its potential for CNF production via mechanical fibrillation. Conditions that maximized CNC yields (60% w/w) were 60% acid concentration, 58 °C, 60 min and 40 mesh particle size. Twenty percent (w/w) of the pulp was degraded under these conditions. On the other hand, conditions that maximized CSR yields (60% w/w) were 54% acid, 45 °C, 90 min and 20 mesh particle size, which also produced 15% CNCs, caused minimal pulp degradation (< 5%) and imparted sufficient surface charge such that CSR was easily microfluidized into CNFs. Therefore, the strong acid hydrolysis process could be tuned to maximize the production of cellulose nanocrystals and nanofibers and obtain two products with different properties and applications through the process optimization.
Streptomyces aminopeptidase P enzymes are proline-specific peptidases that belong to the peptidase M24 family. To evaluate the activity of a commercial Streptomyces aminopeptidase P, named ‘XPO DUET’, we performed three experiments involving degradation of tryptic casein, production of free amino acids from casein hydrolysate, and hydrolysis of synthetic peptides. Using an ion-trap liquid chromatography–mass spectrometry (LC–MS) apparatus, we demonstrate that XPO DUET could degrade FFVAPFPEVFGK, an allergic and bitter peptide, VAPFPEVFGK, and PEVFGK from tryptic casein. All amino acids, except Ala, Asp, Glu, and Tyr, were released in an XPO DUET activity-dependent manner during the hydrolysis of casein hydrolysate. LC–MS analysis also revealed the ability of XPO DUET to completely hydrolyze Phe-Phe-Phe into free Phe. Thus, we confirm that XPO DUET possesses broader specificity than its known activity toward Xaa-Pro peptides. Because XPO DUET is a food-grade peptidase, it is useful in the bioprocessing of protein hydrolysates through its combination with other food-grade peptidases.
For the production of biofuel (bioethanol), enzymatic adsorption onto a lignocellulosic biomass surface is a prior condition for the enzymatic hydrolysis process to occur. Lignocellulosic substances are mainly composed of cellulose, hemicellulose and lignin. The polysaccharide matrix (cellulose and hemicellulose) is capable of producing bioethanol. Therefore, lignin is removed or its concentration is reduced from the adsorption substrates by pretreatments. Selected enzymes are used for the production of reducing sugars from cellulosic materials, which in turn are converted to bioethanol. Adsorption of enzymes onto the substrate surface is a complicated process. A large number of research have been performed on the adsorption process, but little has been done to understand the mechanism of adsorption process. This article reviews the mechanisms of adsorption of enzymes onto the biomass surfaces. A conceptual adsorption mechanism is presented which will fill the gaps in literature and help researchers and industry to use adsorption more efficiently. The process of enzymatic adsorption starts with the reciprocal interplay of enzymes and substrates and ends with the establishment of molecular and cellular binding. The kinetics of an enzymatic reaction is almost the same as that of a characteristic chemical catalytic reaction. The influencing factors discussed in detail are: surface characteristics of the participating materials, the environmental factors, such as the associated flow conditions, temperature, concentration, etc. Pretreatment of lignocellulosic materials and optimum range of shear force and temperature for getting better results of adsorption are recommended.
Halomonas boliviensis is a halophilic microorganism that accumulates poly(3-hydroxybutyrate) (PHB) using different carbons sources when nitrogen is depleted from the culture medium. This work presents an improved production of PHB using an air-lift reactor (ALR) that was fed with a concentrated solution of a carbon source, and was supplemented with an adequate airflow rate.
Simple production media were used to study PHB production by H. boliviensis in an ALR. Glucose was first used as the main carbon source and was fed during the exponential phase of cell growth. The maximum CDW and PHB content were 31.7 g/L and 51 wt%, respectively, when the airflow rate entering the reactor varied between 0.5 and 1.2 L/min. Changing the air inflow to 0.5–0.9 L/min resulted in an improvement in PHB accumulation (62 wt%). A cultivation was performed by using the latter range of airflow rate and feeding glucose only when nitrogen was depleted from the medium; a considerable enhancement in PHB content (72 wt%) and CDW (27 g/L) was achieved under these conditions. Moreover, PHB was also produced using molasses as the main carbon source. Residual cell mass was about the same to that achieved with glucose, however the PHB content (52 wt%) was lower.
PHB production by H. boliviensis in an ALR using a simple medium is possible. CDW and PHB content in H. boliviensis can be improved with respect to batch cultivations previously reported when a carbon source is fed to the reactor. The best strategy for the production of PHB consisted of starting the cultivation in a batch mode while glutamate was present in the medium; glucose should be fed when glutamate is depleted from the medium to keep an excess of the carbon source during the synthesis of PHB.
Diaphorase (DI) has received wide attention as the key anodic enzyme mediating the electron transfer and electric energy generation in enzymatic biofuel cells (EBFCs). Lowering the anodic pH may be a useful strategy for constructing high-performance in EBFCs. However, most DI suffered from the poor activity at low pHs. Therefore, it is necessary to modify the activity and its acidic tolerance to further improve the performance of the EBFC.
This paper attempts to improve the enzyme activity of DI originated from Geobacillus stearothermophilus under acidic conditions through directed evolution. Three rounds of random mutagenesis by error-prone PCR of the GsDI gene followed by high-throughput screening allowed the identification of the mutant 3–8 (H37Q, S73T, F105L, S68T, G61S, D74V) exhibiting a 4- or 7-fold increase in the catalytic activity at pH 5.4 or 4.5 compared to that of the wild type. And the pH stability of mutant 3–8 was significantly better than that of wild type and showed a 1.3 times higher in the stability at pH 5.4. The EBFC anode equipped with 0.5 mg of mutant 3–8 achieved a maximum current of 40 μA at pH 5.4, much higher than that with the same loading of the wild type enzyme.
The GsDI has been improved in the specific activity and pH stability by directed evolution which leads to the improvement of the EBFC performance. Also, the enlarged catalytic channel of mutant and decreased B-factor may be beneficial for the activity and stability. These results suggest that this engineered DI will be a useful candidate for the construction of enhanced EBFCs.
In order to confirm the contribution of delignification to the increase in lignocellulosic cellulose digestibility, several laboratory oxidative pretreatments under mild conditions, including alkaline-hydrogen peroxide (AP), two-step alkaline/peracetic acid (APAA) and sodium chlorite (SC) pretreatments were employed to achieve selective delignification of sugarcane bagasse and retained most of the hemicelluloses (xylan) in the pretreated solids. Four commercial cellulase cocktails were used to test the enzymatic hydrolyzability of pretreated substrates. Results revealed that delignification indeed could greatly improve the final (120 h) cellulose hydrolysis with relatively high final (120 h) glucan conversion (> 90%) by different cellulase cocktails even if the substrates still had a high hemicelluloses content. However, the xylan conversion seemed to be more greatly dependent on the pretreatments and cellulase cocktails used. AP and APAA pretreatments resulted in the disappearance of middle lamella and liberation of cellulose fibers with significant etching, deformation and fracture of cell wall structure. SC pretreatment greatly modified the sugar bagasse surface morphology to make the surface much coarser. The cell wall also underwent serious fracture and deformation with some middle lamella disappearing. However, no significant alteration on the structure of pure cellulose was observed by SC oxidative pretreatment of filter paper. Oxidative pretreatment might also modify lignin structure and surface properties thus greatly reducing the non-specific adsorption of enzymes. The obtained results strongly support the conclusion that delignification under mild pretreatment condition can be very helpful to improve the enzymatic hydrolysis of lignocellulosic cellulose by commercial cellulase cocktails even if the substrates has a high hemicelluloses content.
The effects of cadmium (Cd) contamination on the microbial community structure, soil physicochemistry and heavy metal resistome of a tropical agricultural soil were evaluated in field-moist soil microcosms. A Cd-contaminated agricultural soil (SL5) and an untreated control (SL4) were compared over a period of 5 weeks. Analysis of the physicochemical properties and heavy metals content of the two microcosms revealed a statistically significant decrease in value of the soil physicochemical parameters (P < 0.05) and concentration of heavy metals (Cd, Pb, Cr, Zn, Fe, Cu, Se) content of the agricultural soil in SL5 microcosm. Illumina shotgun sequencing of the DNA extracted from the two microcosms showed the predominance of the phyla, classes, genera and species of Proteobacteria (37.38%), Actinobacteria (35.02%), Prevotella (6.93%), and Conexibacter woesei (8.93%) in SL4, and Proteobacteria (50.50%), Alphaproteobacteria (22.28%), Methylobacterium (9.14%), and Methylobacterium radiotolerans (12,80%) in SL5, respectively. Statistically significant (P < 0.05) difference between the metagenomes was observed at genus and species delineations. Functional annotation of the two metagenomes revealed diverse heavy metal resistome for the uptake, transport, efflux and detoxification of various heavy metals. It also revealed the exclusive detection in SL5 metagenome of members of RND (resistance nodulation division) protein czcCBA efflux system (czcA, czrA, czrB), CDF (cation diffusion facilitator) transporters (czcD), and genes for enzymes that protect the microbial cells against cadmium stress (sodA, sodB, ahpC). The results obtained in this study showed that Cd contamination significantly affects the soil microbial community structure and function, modifies the heavy metal resistome, alters the soil physicochemistry and results in massive loss of some autochthonous members of the community not adapted to the Cd stress.
A novel bioconversion approach of Tunisian wastewater to low-cost Photorhabdus temperata bioinsecticide is presented in this study. Our results showed that when cultured on the food industry wastewater (WS4), P. temperata cells exhibited oral toxicity of about 42%, which is the same as those cultured in complex medium (CM), used as control. Moreover, variants small colony polymorphism (Vsm) of the strain K122 was completely avoided after a prolonged incubation. However, viable but non-culturable (VBNC) state was enhanced with the maximum colony-forming units (CFU) count of 9 × 106 cells/mL obtained after 48 h of incubation in the WS4. According to flow cytometry analysis, almost 100% of P. temperata cells were viable until 48 h of incubation. The appearance of propidium iodide (PI) positively stained cells was observed after a prolonged incubation with a maximum of 17% of damaged cells in WS1. In order to follow the progress of P. temperata fermentation process carried out in industrial wastewater, we established for the first time, the mathematical relationship between total cell counts, CFU counts and oral toxicity of P. temperata strain K122. Indeed, irrespective of the medium used, the relationship between CFU count and total cell count followed a power law. Additionally, when plotting CFU count, or total cell count against toxicity, a semi-log linear relationship was obtained. Our results proved the efficiency of this bioconversion approach to produce bioinsecticide based on the entomopathogenic bacterium P. temperata, with practical benefits in terms of cost production and wastewater management.
Photosynthetic microorganisms are considered excellent feedstock for biofuel production in developing biomass production technologies. A study was conducted to evaluate ethanol production with the sequential enzymatic saccharification and fermentation of Arthrospira platensis (Spirulina) biomass with the metabolically engineered Escherichia coli strain MS04. A. platensis was cultivated semicontinuously in an open raceway pond, and the carbohydrate content was determined to be as high as 40%. The enzymatic saccharification was designed to release the maximum amount of glucose. After 40 h of enzymatic saccharification, 27 g L−1 of monosaccharides was obtained. These slurries were fermented with ethanologenic bacteria, achieving 12.7 g L−1 ethanol after 9 h of fermentation, which corresponds to 92% conversion yield of the glucose content in the hydrolysate, 0.13 g of ethanol per 1 g of Spirulina biomass and a volumetric productivity of 1.4 g of ethanol L−1 h−1. Therefore, we conclude that it is possible, in a short time, to obtain a high ethanol yield corresponding to 160 L per ton of dry biomass with a high productivity.
The fungus–substrate interaction on the antioxidant activity of a solid state fermentation system (SSFS) was investigated employing two basidiomycete fungi: Pleurotus columbinus and P. floridanus and two ascomycetes: Aspergillus fumigatus and Paecilomyces variotii on powdered peels of banana, pomegranate and orange, empty pea pods and rice straw. The oven-dried substrates were moistened at 90% water holding capacity, inoculated with the test fungi and incubated at 25 °C for appropriate time. Culture extracts were tested for the enzymatic and non-enzymatic antioxidant activity. The effect of substrate on the antioxidant activity of the SSFS was stronger than that of the fungal species. Peroxidase (POX) activity was higher in the basidiomycetes than ascomycetes and achieved its maximum in P. floridanus versus complete absence in A. fumigatus. By contrast, catalase (CAT) activity was higher in the ascomycetes, particularly P. variotii on banana peel, than in the basidiomycetes. Phenolics and flavonoids were highest in pomegranate peels but lowest in banana peels and rice straw, and they were subjected to severe consumption by the basidiomycetes versus mild production by the ascomycetes. The reducing power (RP) and DPPH scavenging activity were higher in the peels of pomegranate, orange and banana relative to rice straw and empty pea pods, and the fungal effect was limited and independent of the fungal taxonomic group. Orange peel is the appropriate substrate for production of fungal CAT and POX, which activities were mutually exclusive; but pomegranate peel is more suitable for production of phenolics and flavonoids.
Lignin’s valorization plays a critical role in refining the bioresource. Considering that the β-aryl ether linkage (β-O-4 bond) accounts for 50–70% of lignin chemical linkage between aromatic rings, the hydrolase of lignin β-aryl ether linkage, especially the β-etherase, provided a promising way for the lignin depolymerization and valorization. As a result, it is essential to develop the effective high-throughput methods for screening the mutant library of β-etherase from directed evolution.
Based on the enzymatic mechanism of β-O-4 bond’s cleavage by β-etherase, the LigF was selected as the model to study high-throughput method by GSH assay for screening the mutant library of β-etherase from directed evolution. After the primary study with purified LigF and cell lysate, the GSH assay was used to screen mutant library of β-etherase. The study on screening the mutant library with about 600 colonies indicated that the selected transformants all have one or two mutated sites in the gene sequence of LigF, and the activities from GSH assay of most selected transformants were the same as their activities from HPLC assay.
The results from the high-throughput screening of mutant library demonstrated that GSH assay could be applied to screen β-etherase mutant from directed evolution.
The study was designed to evaluate the production of auxin by eukaryotic unicellular organism Pichia fermentans. Different media formulations were used for the production of indole-3-acetic acid (IAA) under broth and submerged conditions. Wheat straw-based production medium was formulated and optimized using statistical approach. The IAA production was significantly enhanced by nine folds, when the wheat straw was pretreated with Phanerochaete chrysosporium (150 µg/ml) as compared to untreated wheat straw (16.44 µg/ml). Partial purification of IAA was carried out by silica gel column chromatography and further confirmed by high-performance liquid chromatography. Exogenous application of crude and partially purified IAA positively influenced the Vigna radiata seedling growth. The number of lateral roots in the growing seedlings was significantly higher as compared to the control seeds. Thus, the present findings point towards an efficient production of plant hormone by yeast and white rot fungus using abundantly available wheat straw, which may lead to the development of cost-effective production of such metabolites and their further use in agricultural field to reduce the negative impact of chemical fertilizers.
Recent advances in metabolite quantification and identification have enabled new research into the detection and control of titer inhibitors and promoters. This paper presents a bigdata analytics study to identify both inhibitors and promoters using multivariate data analysis of metabolomics data. By applying multi-way partial least squares (PLS) model to metabolite data from four fed-batch bioreactor conditions where feed formulation and selection agent concentrations varied, metabolites which exhibited the most significant impact on titer during cultivation were ranked from highest to lowest. The model outputs were then constrained to reduce the number of statistically relevant inhibitors or promoters to the top ten, which were used to conduct metabolic pathway analysis. Furthermore, a method is presented for identifying amino acids that prevent the accumulation of the inhibitors and/or enhance the formation of promoters during production. Finally, the metabolomics and pathway analysis results were integrated and validated with transcriptomics data to characterize metabolic changes occurring among different growth conditions. From these results, new feeding strategies were implemented which resulted in increased fed-batch production titer. Methodology from this work could be applied to future process optimization strategies for biotherapeutic production.
Monacolin J (MJ) is a key intermediate for the synthesis of cholesterol-lowering drug simvastatin. Current industrial production of MJ involves complicated chemical hydrolysis of microbial fermented lovastatin. Recently, heterologous production of MJ has been achieved in yeast and bacteria, but the resulting metabolic stress and excessive accumulation of the compound adversely affect cell activity.
Five genes, tapA, stapA, slovI, smokI and smlcE, coding for fungal statin pump proteins were expressed in an MJ producing yeast strain, Komagataella phaffii J#9. Overexpression of these genes facilitated MJ production. Among them, tapA from Aspergillus terreus highly improved MJ production and led to a titer increase of 108%. Exogenous MJ feeding study on an MJ non-producing strain GS-PGAP-TapA was then performed, and the results illustrated tough entry of MJ into cells and possible efflux action of TapA. Further, intracellular and extracellular MJ levels of J#9 and J#9-TapA were analyzed. The extracellular MJ level of J#9-TapA increased faster, but its intracellular MJ percentage kept lower as compared to J#9. The results proved that TapA effectively excreted MJ from cells. Then functions of TapA were evaluated in a high-production bioreactor fermentation. Differently, TapA expression caused a low MJ titer but high intracellular MJ accumulation in J#9-TapA compared with J#9.
Statin pump proteins improved MJ production in K. phaffii in a shake flask. Exogenous MJ feeding and endogenous MJ producing experiments demonstrated the efflux function of TapA. TapA improved MJ production at low MJ levels in a shake flask, but decreased it at high MJ levels in a bioreactor. This finding is useful for statin pump improvement and metabolic engineering for statin bioproduction.
Hyperlipidemia is involved in serious cardiovascular disease, however, synthetic drugs to reduce lipid contents in blood stream have been found to induce serious side effects. In the current study, we compared anti-oxidant and anti-hyperlipidemic effect of Paecilomyces japonica (PJ), Cordyceps militaris (CM) and cordycepin-rich Cordyceps militaris (CMα) in rats induced alcoholic hyperlipidemia (AIH) and oxidative stress. The experimental groups were divided in N (water), C (30% alcohol), PJ (30% alcohol + 3% PJ powder), CM (30% alcohol + 3% CM powder), CMα (30% alcohol + 3% CMα powder) and SM [30% alcohol + 0.1% silymarin (SM)]. Compared to C group, supplementation of PJ, CM, CMα and SM slightly alleviated the increased weight ratio of liver and kidney in the alcohol-treated rats. In addition, a significant or slight reduction was identified in total lipid, total cholesterol and HDL-cholesterol levels in the rats receiving PJ, CM and CMα as compared with C group. Administration of PJ, CM and CMα also blocked alcohol-induced lipid peroxidation via a decrease of malondialdehyde (MDA), and activated anti-oxidant enzyme, glutathione (GSH), in serum and various organ tissues. Overall, cordycepin-rich CMα showed highest anti-oxidant and anti-hyperlipidemia effect under chronic alcoholic damage. Our results indicate that CMα might be useful in inhibiting the oxidation and hyperlipidemia in alcohol-induced hepatic disease possibly because of potential anti-oxidative and anti-hyperlipidemic activities of cordycepin.
The presence of heavy metals in polluted water is known not only to cause severe harm to marine organisms, but also to terrestrial plants and animals including human beings. This research applied low-cost and environmentally benign adsorbent primed from waste orange peel (OP) for the removal of Cd(II) ions from aqueous solution via batch adsorption process. The surface properties of the orange peel powder were studied using scanning electron microscopy (SEM), X-ray spectroscopy (XRD) and Fourier transform infrared spectroscopy (FT-IR). Operational conditions like temperature, contact time, sorbent dosage, solution pH and initial adsorbate concentration were investigated. The utmost uptake of Cd(II) ion was obtained at a contact time of 120 min, initial metal concentration of 240 mg/L, adsorbent dosage of 0.04 g/L, temperature of 45 °C and solution pH of 5.5. Equilibrium results showed that the orange peel adsorbent has an adsorption capacity of 128.23 mg/g as obtained from the Langmuir isotherm. The adsorption kinetics data followed a pseudo-first-order kinetic model with correlation coefficient (R2) > 0.9 and low standard % error values. The adsorption process was found to be endothermic in nature with enthalpy of 0.0046 kJ mol−1 and entropy of-636.865 Jmol−1K−1. Results from the optimization study indicated that higher adsorbent dosage and lower Cd(II) ion concentration increased the percentage of Cd(II) ion removal. Thus, orange peel could be used in the removal of Cd(II) ion from aqueous solutions.
The grand challenge now and in the near future for the pharmaceutical industry is how to efficiently improve R&D productivity. Currently, the approval rate of the entire clinical drug development process is extremely low, and the high attrition in the phase I clinical trial is up to 95%; 67% and 33% of all drugs that enter Phase II and Phase III clinical trials fail to transit into the next stage, respectively. To achieve a higher success rate in clinical trials, developing efficient drug screening method based on more in vivo like tumor tissue is an urgent need to predict the toxicity and efficacy of candidate drugs. In comparison to 2D planar tumor model, the 3D multicellular tumor spheroid (MTS) can better simulate the spatial structure, hypoxia and nutrient gradient, extracellular matrix (ECM) deposition and drug resistance mechanism of tumor in vivo. Thus, such model can be applied for high-throughput drug screening and evaluation, and also can be utilized to initiate a series of fundamental research areas regarding oncogenesis, tumor progression and invasion, pharmacokinetics, drug metabolism, gene therapy and immune mechanism. This review article discusses the abnormal metabolism of cancer cells and highlights the potential role of MTSs as being used as efficient preclinical models. Also, the key features and preparation protocols of MTSs as well as the tools and techniques used for their analysis were summarized and the application of 3D tumor spheroid in specific drug screening and in the elucidation of drug resistance mechanism was also provided. Despite the great knowledge gap within biological sciences and bioengineering, the grand blueprint for adaptable stirred-tank culture strategies for large-scale production of MTSs is envisioned.
Current research in industrial microbiology and biotechnology focuses on the production of biodegradable microbial polymers as an environmentally friendly alternative to still dominant fossil-based plastics. Microbial polymers have an extensive biotechnological potential and are already widely used in a variety of fields ranging from medicine to technology. However, their increase in production and wider use is hampered by the high cost of raw materials and therefore requires a focus on cheaper inputs, including dairy by-products and waste such as cheese whey (CW). This is an environmentally unfriendly by-product of milk processing and reducing it would also reduce the risk of environmental pollution. This review summarises current knowledge on the use of CW and derived products to obtain commercially important microbial polymers, including information about producer cultures, fermentation techniques and methods used, composition of culture medium, cultivation conditions and productivity of bioprocesses. The main methods and applications of cheese whey pre-treatment are also summarised.
Cutinases could degrade insoluble polyester, including natural cutin and synthetic plastic. However, their turnover efficiency for polyester remains too low for industrial application. Herein, we report the 1.54-Å resolution X-ray crystal structure of a cutinase from Thermobifida fusca and modeling structure in complex with a cutin mimic oligo-polyester C24H42O8. These efforts subsequently guided our design of cutinase variants with less bulky residues in the vicinity of the substrate binding site. The L90A and I213A variants exhibit increased hydrolysis activity (5- and 2.4-fold, respectively) toward cutin and also showed enhanced cotton scouring efficiency compared with the wild-type enzyme.
We report the high production of stilbenes, including resveratrol and viniferin, in grapevine (Vitis labruscana L.) cell cultures through elicitation with methyl jasmonate (MeJA) and stevioside (STE). Methyl-β-cyclodextrin (MeβCD) is widely used as a solubilizer for resveratrol production. For the first time, we used STE as a solubilizer for stilbene production in plant cell cultures. MeJA was most effective elicitor in activating VvSTS expression and stimulating stilbene biosynthesis in grapevine cell cultures. The maximum concentration of δ-viniferin (892.2 mg/L) production with a small amount of trans-resveratrol (12.2 mg/L) was observed in the culture medium by co-treatment of cells with MeJA and STE, whereas the highest level of trans-resveratrol (371.9 mg/L) with a slight amount of δ-viniferin (11.5 mg/L) was accumulated in the culture medium of cells treated with MeJA and MeβCD. However, neither trans-resveratrol nor δ-viniferin were significantly elevated within the cells by the applications. Notably, predominant production of δ-viniferin and trans-resveratrol was observed in shake and static flask culture medium, respectively, by co-treatment of MeJA and STE. Furthermore, stilbene compounds of resveratrol, ε-viniferin, and δ-viniferin were mainly produced in a 3-L bioreactor culture following elicitation of cells with MeJA and STE. These results provide new strategies for conditional, high-level production of resveratrol and viniferin in cell cultures by utilizing the solubilizing properties of STE or MeβCD.
Microalgae is one of the major sources of natural compounds with antimicrobial activity. The metabolite profiling of the extracts could identify the bioactive compounds based on methanol (MET), ethanol (ETH), chloroform (CHL), hexane (HEX) and water (W) solvent systems. The microalgal crude extracts in co-application with silver nanoparticles (AgNPs) had enhanced antimicrobial activity with potential to overcome the global problem of microbial antibiotic resistance.
Chlorella sp. exhibited the highest lipid, N. oculata the highest total saturated fatty acids (TSFA), and T. suecica the highest mono-unsaturated (MUFA) and poly-unsaturated fatty acids (PUFA). The highest carbohydrate, protein and total phenolics contents (TPCs) were attained by N. oculata. The highest total flavonoids contents (TFCs), and chlorophyll a and b were in T. suecica, while comparable level of carotenoids were found in all species. For high-performance thin-layer chromatography (HPTLC) analyses, the eicosapentaenoic acid (EPA) with high peaks were detected in T. suecica-HEX and N. oculata-CHL; and β-carotene in Chlorella sp.-ETH. The gas chromatography–mass spectrometry (GC–MS) analyses showed high 13-docosenamide (Z)- in T. suecica-HEX; phytol in N. oculata-HEX; and neophytadiene in Chlorella sp.-ETH. The AgNPs–MCEs–MET and HEX at the 1.5:1 ratios exhibited strong activities against Bacillus subtilis, Streptococcus uberis, and Salmonella sp.; and the AgNPs–T. suecica-HEX and MET and AgNPs–Chlorella sp.-HEX at the 1.5:1 ratios exhibited activities against Klebsiella pneumoniae.
Different bioactive components were detected in the MCEs based on the HPTLC and GC–MS analyses. Significant antimicrobial activities against the pathogenic microbes were demonstrated by the synergistic effects of the MCEs in co-application with the AgNPs. This could be beneficial in the fight against sensitive and multidrug-resistant bacteria.
Sorghum has been used for brewing European beers but its malt generally lower beer yields and alcohol contents. The aim of this research was to produce lager beers using worts from sorghum malted with and without Aspergillus oryzae inoculation. Worts adjusted to 15° Plato from the sorghum malt inoculated with 1% A. oryzae yielded 21.5% and 5% more volume compared to sorghum malt and barley malt worts, respectively. The main fermentable carbohydrate in all worts was maltose. Glucose was present in higher amounts in both sorghum worts compared to barley malt worts. Sorghum–A. oryzae beer had similar specific gravity and alcohol compared to the barley malt beer. Sorghum–A. oryzae beer contained lower amounts of hydrogen sulfide, methanethiol, butanedione, and pentanedione compared to barley malt beer. Sorghum–A. oryzae lager beer had similar yield and alcohol content compared to the barley malt beer but differed in color, key volatiles and aromatic compounds.
Most cellulose-based materials’ manufacturing processes include processing this biopolymer in an aqueous medium. Sorption properties depend on cellulose supramolecular structure and nature of its change during moistening. Plenty of researchers’ efforts have been directed to the development of scientifically sound and commercially reliable processes over the past decade for the cellulose fibers’ dispersion in an aqueous medium. Therefore, it needs a more detailed study of the cellulose–water system components’ interaction. This study presents the supramolecular structure and sorption properties of native cotton cellulose research results obtained by 1H NMR relaxation, spectroscopy and sorption measurements. Hydrophilic properties of cellulose as an adsorbent are characterized, taking into account a porous system between its structural elements. We examine in detail water adsorption on the active surface of cellulose Iβ. We also demonstrate the approach for determining the entropy change in the first two layers of adsorbed water and estimate this value increased during adsorption. Cellulose moistening is accompanied by the decomposition of macrofibrils into microfibrils and is manifested in a crystallinity decrease and a specific surface area growth.
Any type of biomass can be used as substrate for biogas production, but the performance of the biodigestion depends on the composition of the feed, and no direct extrapolation of the yield of the process from one substrate to another can be made. In this work, the performance of a bench-scale anaerobic biodigester of 93 L installed at ambient conditions is studied. The biodigester was set up in a region where temperature varies significantly during the year, and was operated under semi-batch conditions with non-thermal control for 16 months with a feed of rabbit manure and ground sorghum grains. To our knowledge, this is the first time the co-digestion of rabbit manure with sorghum grains is considered. To evaluate the biodigestion performance, critical operational variables (pH, temperature, biogas flowrate) were monitored, and composition of substrate, digestate and produced biogas was determined. Moreover, the following variables were quantified: (a) the theoretical methane potential, (b) the specific methane yield and (c) the degree of degradation of the substrate. A 1-D non-stationary model was formulated and validated with experimental data in order to analyze, in a theoretical form, the impact of incorporating thermal insulation to the unit. The results show that is it possible to produce biogas in a bench-scale biodigester, with a novel feed of rabbit manure and ground sorghum grains, in a region with significant temperature changes along the year. Moreover, it is shown that the 1-D model constitutes a useful tool for the design or improvement of biodigesters regarding the insulation system and the warming policies.
Microalgae Nannochloropsis sp. is a widely recognized renewable biodiesel feedstock. The ability of this microalgae to absorb CO2 constitutes an added value toward reducing global warming. However, the process of optimizing its growth still involves many challenges. Photoinhibition, which takes places during microalgae cultivation when using continuous lighting, constitutes an unresolved problem. Therefore, the optimum light/dark cycle method is considered necessary. The experiments were conducted using a designed, tubular airlift photobioreactor and blue, energy-saving, light-emitting diode (LED) lights for the purpose of internal illumination. We observed that a 45:15 min (light:dark) cycle increased the production of Nannochloropsis sp. biomass significantly, with a cell density, wet weight, and lipid content of 17 × 106 cell/ml, 7.11 g, and 10.1% dry weight, respectively. By using the blue LED lights, our designed, airlift photobioreactor increased cell growth by 70% compared to the growth of Nannochloropsis sp. in nature and produced 61 times higher lipid content compared to Nannochloropsis sp. that is exposed to natural light.
Multiple wastes’ co-digestion is one of the alternatives for improved anaerobic digestion (AD) process of industrial and municipal wastes. The present work investigated the influence of fruit–vegetable solid waste (FVW) addition as a co-substrate on the performance of AD of abattoir wastewater (AWW). The co-digestion was done at a lab-scale-based experiment under mesophilic condition using a two-phase anaerobic sequencing batch reactor without agitation. It was tested at different mixing ratios (100%AWW; 75%AWW:25%FVW; 50%AWW:50%FVW; 25%AWW:75%FVW; 100%FVW) with the intention of looking for the best mixing ratio with the best performance. It was fed on a semi-continuous basis and operated for 18 days (d) total retention time (HRT): 3 days for the acidogenesis reactor and 15 days for methanogenesis reactor. The addition of FVW enhanced biogas yield and VS removal by 70.26% and 57.11%, respectively, at optimum mixing ratio. Moreover, to some extent improvement of AD process stability verified by the decreased TVFA:TAlk ratio and free ammonia nitrogen was observed upon progressive addition of FVW. Finally, this co-digestion process should further be studied for its performance at different HRTs with agitation.
Oligosaccharides have important therapeutic applications. A useful route for oligosaccharides synthesis is reverse hydrolysis by β-glucosidase. However, the low conversion efficiency of disaccharides from monosaccharides limits its large-scale production because the equilibrium is biased in the direction of hydrolysis. Based on the analysis of the docking results, we hypothesized that the hydropathy index of key amino acid residues in the catalytic site is closely related with disaccharide synthesis and more hydrophilic residues located in the catalytic site would enhance reverse hydrolysis activity. In this study, positive variants TrCel1bI177S, TrCel1bI177S/I174S, and TrCel1bI177S/I174S/W173H, and one negative variant TrCel1bN240I were designed according to the Hydropathy Index For Enzyme Activity (HIFEA) strategy. The reverse hydrolysis with TrCel1bI177S/I174S/W173H was accelerated and then the maximum total production (195.8 mg/mL/mg enzyme) of the synthesized disaccharides was increased by 3.5-fold compared to that of wild type. On the contrary, TrCel1bN240I lost reverse hydrolysis activity. The results demonstrate that the average hydropathy index of the key amino acid residues in the catalytic site of TrCel1b is an important factor for the synthesis of laminaribiose, sophorose, and cellobiose. The HIFEA strategy provides a new perspective for the rational design of β-glucosidases used for the synthesis of oligosaccharides.
Advances in biochemical and molecular manipulation have led to increased biomass productivity and oil accumulation in the microalgae C. reinhardtii. However, scalable processes for the recovery of oil and other valuable biomolecules, such as protein, from C. reinhardtii are scarce. The use of aqueous enzymatic extraction, a non-solvent and environmentally friendly bioproduct recovery method, provides an opportunity to design an integrated process for oil and protein fractionation to reduce bioenergy and bioproducts costs. Based on the mechanistic understanding of biomolecule distribution and compartmentalization, an aqueous enzymatic treatment for the release of internally stored lipid bodies was designed. Application of a C. reinhardtii-produced protease, autolysin, for lysis of the microalgae cell wall was followed by a secondary treatment with trypsin for chloroplast disruption and lipid body release. Protein recovery after the primary treatment with autolysin indicated a 50.1 ± 4.2% release of total soluble protein and localization of lipid bodies still in the chloroplast. The development of a secondary enzyme treatment (trypsin) for chloroplast and lipid body lysis demonstrated a high percent of remaining lipids (73 ± 7%) released into the supernatant. The results indicate that the application of an enzymatic treatment scheme for protein and oil recovery is a promising alternative to traditional extraction processes.
Arthrospira platensis is a cyanobacterium that is of great biotechnological interest, particularly for the food industry, as it possesses a high content of proteins, pigments, lipids and carbohydrates. Cyanobacteria produce extracellular polymeric substances (EPS), which are co-products of secondary metabolism that present thickening or gelling properties. A 3-level factorial design was used to study the combined effect of different nitrate concentrations and photon flux density (PFD) values to evaluate the biomass and EPS production of A. platensis. The best result in terms of biomass production was obtained under condition 6 (2 g L−1 NaNO3 and 600 μE m−2 s−1) yielding a concentration of 1.292 g L−1. However, condition 1 (0.25 g L−1 NaNO3 and 200 μE m−2 s−1) produced the greatest EPS yield (111 mg g−1), followed by condition 9 (2 g L−1 NaNO3 and 1000 μE m−2 s−1). FTIR analyses of EPS samples indicated the presence of carboxylate and sulfate functional groups, and rheological studies of the EPS at 5 and 10 g L−1 revealed a dilute solution behavior.
There is a significant interest in converting eggshells into value-added products. Therefore, the goal of this research is to synthesize and study iron-impregnated eggshells as a catalyst for partial oxidation of methane. The objectives of this research were to test the effects of iron loading, flow rate, oxygen concentration, and temperature on methane oxidation. The catalysts were synthesized using ferric chloride hexahydrate at various loadings and tested in a heated stainless-steel reactor under different experimental conditions. The reaction products included C2–C7 hydrocarbons, carbon monoxide, and carbon dioxide depending on the reaction conditions. Results indicated that iron loading beyond 5 wt% caused a decrease in methane conversion. A decrease in oxygen concentration enhanced methane conversion with a substantial drop in the production of CO2. Besides, an increase in temperature resulted in a decrease in methane conversion with a simultaneous increase in the production of CO2 via overoxidation. The reusability experiments indicated that the catalyst was active for four reaction cycles. Our results indicate that eggshells can be used as catalyst support for methane partial oxidation and can simultaneously solve the waste disposal problems faced by the poultry industry.
An amendment to this paper has been published and can be accessed via the original article.
Pretreatment of keratinous residues using an ultrasonic reaction system provides greater enzymatic production in less time. This is a promising technology for measuring enzyme activity and microwave processes. In the present work, an ultrasonic probe reaction system was used to evaluate the potential of swine hair pretreatment. The pretreated material was submerged with non-pretreated residues for 9 days to obtain the enzyme. Enzyme activity was measured in the extracts obtained using the ultrasonic probe, ultrasonic bath, and microwave. We also used the enzymatic concentration technique with NaCl and acetone. Homemade enzymatic extracts were evaluated for their ability to degrade swine hair and chicken feathers by comparing them with the activities commercial enzymes. Macrobeads gave greater energy dissipation in less time, providing greater enzyme activity (50.8 U/mL over 3 days). In terms of waste degradation, non-pretreated swine hair was more promising. The ultrasonic probe reaction system had the potential to evaluate increased enzyme activity (38.4% relative activity) and the enzyme concentration increased activity by 53.5%. The homemade enzymatic extract showed promise for degradation of keratinous residues.
Natural lignocellulosic fibres (NLF) extracted from different industrial crops (like cotton, hemp, flax, and canola) have taken a growing share of the overall global use of natural fibres required for manufacturing consumer apparels and textile substrate. The attributes of these constituent NLF determine the end product (textiles) performance and function. Structural and microscopic studies have highlighted the key behaviors of these NLF and understanding these behaviors is essential to regulate their industrial production, engineering applications, and harness their benefits. Breakthrough scientific successes have demonstrated textile fibre properties and significantly different mechanical and structural behavioral patterns related to different cultivars of NLF, but a broader agenda is needed to study these behaviors. Influence of key fibre attributes of NLF and properties of different cultivars on the performance of textiles are defined in this review. A likelihood analysis using scattergram and Pearson’s correlation followed by a two-dimensional principal component analysis (PCA) to single-out key properties explain the variations and investigate the probabilities of any cluster of similar fibre profiles. Finally, a Weibull distribution determined probabilistic breaking tenacities of different fibres after statistical analysis of more than 60 (N > 60) cultivars of cotton, canola, flax, and hemp fibres.
The “alperujo” is a waste from the olive oil industry with great potential for valorization. It has a high organic load, with the presence of valuable compounds such as biophenols and sugars. The use of this waste can be thought of as a biorefinery from which different compounds of high added value can be obtained, whether they are present in the “alperujo” such as biophenols or can be generated from the “alperujo”. Therefore, the production of carotenoids by Rhodotorula mucilaginosa was evaluated using the liquid fraction of ‘alperujo’ (Alperujo Water, AW) or an aqueous extract (AE) of “alperujo” at different concentrations (5, 10, 20 and 30% w/V) as substrates. The AEs had an acidic pH, a total sugar concentration ranging from 1.6 to 7.6 g/L, a polyphenols content from 0.4 to 2.9 g/L and a significant amount of proteins (0.5–3 g/L). AW is similar in composition as 30% AE, but with a higher amount of total sugars. Rh. mucilaginosa was able to grow at the different mediums with consumption of glucose and fructose, a reduction in protein content and alkalinization of the medium. Maximum total carotenoid production (7.3 ± 0.6 mg/L) was achieved at AW, while the specific production was higher when the yeast grew at AW or at 30% AE (0.78 ± 0.06 and 0.73 ± 0.10 mg/g of biomass, respectively). Torulene and torularhodin were the main carotenoids produced. Polyphenol content did not change; thus, it is still possible to recover these compounds after producing carotenoids. These results demonstrate the feasibility of using alperujo-based mediums as cheap substrates to produce torularhodin and torulene and to include this bioprocess as a step in an integral approach for alperujo valorization.
Biodecolorization by microorganisms is a potential treatment technique because they seem to be environmentally safe. In the present study, the decolorization and detoxification of cotton blue, crystal violet, malachite green and methyl violet by endophytic fungi were investigated. Preliminary screening result indicated that SWUSI4, identified as Bjerkandera adusta, demonstrated the best decolorization for the four TPM dyes within 14 days. Furthermore, optimization result demonstrated the decolorization rate could reach above 90% at 24 h by live cells of isolate SWUSI4 when 4 g biomass was added into 100-mL dyes solution with the concentration 50 mg/L and shaking (150 rpm) conditions. Moreover, decolorization mechanism analysis shows that the decolorization was caused by the isolate SWUSI4 that mainly includes both absorption of biomass and/or degradation of enzymes. Biosorption of dyes was attributed to binding to hydroxyl, amino, phosphoryl alkane, and ester–lipids groups based on Fourier transform infrared (FTIR) analyses. The biodegradation potential of SWUSI4 was further suggested by the change of peaks in the ultraviolet–visible (UV–vis) spectra and detection of manganese peroxidase and lignin peroxidase activities. Finally, the phytotoxicity test confirmed that the toxicity of TPM dyes after treatment with SWUSI4 was significantly lower than that before treatment. These results indicate that an endophytic SWUSI4 could be used as a potential TPM dyes adsorption and degradation agent, thus facilitating the study of the plant–endophyte symbiosis in the bioremediation processes.
Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. Among all, Escherichia coli possess advantages of fast growth rate, easy for culture and comprehensive elements for genetic engineering. As E. coli W3110 owns the benefits of more heat shock proteins and higher tolerance to toxic chemicals, further execution of T7-based system in W3110 as cell factory is a conceivable strategy.
Three novel W3110 strains, i.e., W3110:IL5, W3110::L5 and W3110::pI, were accomplished by chromosome-equipped T7RNAP. At first, the LacZ and T7RNAP with isopropyl-β-D-thiogalactopyranoside (IPTG) induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), carbonic anhydrase (CA) for carbon dioxide uptake and lysine decarboxylase (CadA) to produce a toxic chemical cadaverine (DAP). All the proteins showed better expression in W3110::L5 and W3110::pI, respectively. As a result, the highest cadaverine production of 36.9 g/L, lysine consumption of 43.8 g/L and up to 100% yield were obtained in W3110::pI(−) with plasmid pSU-T7-CadA constitutively.
Effect of IPTG and lacI/lacO regulator has been investigated in three chromosome-based T7RNAP E. coli strains. The newly engineered W3110 strains possessed similar protein expression compared to commercial BL21(DE3). Furthermore, W3110::pI displays higher production of sfGFP, CA and CadA, due to it having the highest sensitivity to IPTG, thus it represents the greatest potential as a cell factory.
Agroresidues have continued to gain preference over conventional carbon sources for microbial enzyme production due to the low price and abundance in the environment. Therefore, this study aimed at improving peroxidase yield by Bacillus sp. MABINYA-1 (BMAB-1) using agroresidues under submerged fermentation. The culture parameters that support maximum peroxidase yield by BMAB-1 was initially determined and the results showed that peroxidase activity expression was optimum at pH 5, 30 °C and 150 rpm while veratryl alcohol and ammonium sulphate served as the best peroxidase-inducer and inorganic nitrogen source, respectively. BMAB-1 exhibited maximum peroxidase expression (17.50 ± 0.10 U/mg) at 72 h using kraft lignin liquid medium (KLLM) under the optimized culture conditions. Upon utilization of selected agroresidues (sawdust, wheat straw and maize stover) as sole carbon sources by BMAB-1 in the fermentation process, peroxidase activity was significantly enhanced when compared with glucose (14.91 ± 0.31 U/mg) and kraft lignin (17.50 ± 0.10 U/mg). Sawdust produced the highest peroxidase yield (47.14 ± 0.41 U/mg), followed by maize stover (37.09 ± 0.00 U/mg) while wheat straw yielded the lowest peroxidase specific activity (21.65 ± 0.35 U/mg). This indicates that utilization of sawdust by BMAB-1 resulted in 3.2- and 2.7-fold increase in peroxidase activity expression as compared to glucose and kraft lignin, respectively. The aptitude of BMAB-1 to utilize agroresidues would reduce the cost of peroxidase production by the bacteria since the substrates are cheaper than the conventional carbon sources and are, as well, more readily available.
Hexanol–butanol–ethanol fermentation from syngas by Clostridium carboxidivorans P7 is a promising route for biofuel production. However, bacterial agglomeration in the culture of 37 °C severely hampers the accumulation of biomass and products. To investigate the effect of culture temperature on biomass growth and higher-alcohol production, C. carboxidivorans P7 was cultivated at both constant and two-step temperatures in the range from 25 to 37 °C. Meanwhile, Tween-80 and saponin were screened out from eight surfactants to alleviate agglomeration at 37 °C. The results showed that enhanced higher-alcohol production was contributed mainly by the application of two-step temperature culture rather than the addition of anti-agglomeration surfactants. Furthermore, comparative transcriptome revealed that although 37 °C promoted high expression of genes involved in the Wood–Ljungdahl pathway, genes encoding enzymes catalyzing acyl-condensation reactions associated with higher-alcohol production were highly expressed at 25 °C. This study gained greater insight into temperature-effect mechanism on syngas fermentation by C. carboxidivorans P7.
Cyanobacteria and Spirulina produce C-phycocyanin (CPC), a water soluble protein associated pigment, which is extensively used in food and pharmaceutical industries. Other therapeutic proteins might exist in microalgal cells, of which there is limited knowledge. Such proteins/peptides with antibiotic properties are crucial due to the emergence of multi-drug resistant pathogens. In addition, the native expression levels of such disease resistant proteins are low, hindering further investigation. Thus, screening and overexpression of such novel proteins is urgent and important. In this study, a protein which was identified as a putative disease resistance protein (DRP) in the mixture of Spirulina product has been explored for the first time. To improve protein expression, DRP was cloned in the pET system, co-transformed with pRARE plasmid for codon optimization and was significantly overexpressed in E. coli BL21(DE3) under induction with isopropyl-β-d-1-thiogalactopyranoside (IPTG). Furthermore, soluble DRP exhibited intense antimicrobial activity against predominant pathogens, and an inhibition zone of 1.59 to 1.74 cm was obtained for E. coli. At a concentration 4 mg/mL, DRP significantly elevated the growth of L. rhamnosus ZY up to twofold showing probable prebiotic activities. Moreover, DRP showed potential as an effective antioxidant, and the scavenging ability for ROS was in the order of hydroxyl > DPPH > superoxide radicals. A putative disease resistance protein (DRP) has been identified, sequenced, cloned and over-expressed in E. coli as a functional protein. Thus expressed DRP showed potential anti-microbial and antioxidant properties, with promising therapeutic applications.
By seeding fungus on top of industry residues, a mycelium can grow and form a compact network structure; however, it may not develop due to lack of optimal nutrients from the substrate. Consequently, peach-palm residues can be a potential alternative; so, to test this hypothesis, this work evaluates the effect of peach-palm residues as substrate for the growth of mycelium based on Lentinula edodes. They were also supplemented with cassava bran and various sources of nitrogen-ammonium sulphate, potassium nitrate, and soy flour—to analyse its effects on its physico-chemical, enzymatic activities, and thermal and mechanical properties of the final composite at 12 and 20 days of cultivation. This mycelium was able to grow at optimum source treatment conditions, which depends on the ratio of Carbon to Nitrogen, within only 12 days of inoculation. Furthermore, the enzyme activities directly correlate with the mycelium growth with optimum conditions of pH, water activity, and moisture for L. edodes to grow having lower enzyme activities for a well-developed composite; whereas higher activities were seen for a weakly developed material, and this material demonstrates mechanical and thermal properties similar to common mycelium-based composites. Therefore, this work demonstrates that peach-palm residues can be a potential alternative for mycelium-based composite.
Dioxins are the most toxic known environmental pollutants and are mainly formed by human activities. Due to their structural stability, dioxins persist for extended periods and can be transported over long distances from their emission sources. Thus, dioxins can be accumulated to considerable levels in both human and animal food chains. Along with sediments, soils are considered the most important reservoirs of dioxins. Soil microorganisms are therefore highly exposed to dioxins, leading to a range of biological responses that can impact the diversity, genetics and functional of such microbial communities. Dioxins are very hydrophobic with a high affinity to lipidic macromolecules in exposed organisms, including microbes. This review summarizes the genetic, molecular and biochemical impacts of dioxins on the lipid metabolism of soil microbial communities and especially examines modifications in the composition and architecture of cell membranes. This will provide a useful scientific benchmark for future attempts at soil ecological risk assessment, as well as in identifying potential dioxin-specific-responsive lipid biomarkers. Finally, potential uses of lipid-sequestering microorganisms as a part of biotechnological approaches to the bio-management of environmental contamination with dioxins are discussed.
In Tunisia, there are crucial challenges facing both urban and rural areas, the most prominent of which are the production of organic waste, the need for waste treatment, the demand for water and energy and the need for a circular economy. To this end, the study was designed to develop a technical concept on closed cycle ‘biowaste to bioenergy’ treating, basically food waste (FW) through combined biological processes. In this approach, the generated digestate from FW anaerobic reactors was used successfully as a moisturizing agent for FW in-vessel composting. Four types of digestate were examined to be used as moisturizing agent (MA). The selection of the appropriate MA was achieved based on technical criteria; moisture content (MC), C:N ratio and heavy metals concentrations. The findings showed that the digestate obtained from anaerobic co-digestion of food waste and wheat straw (D1) was the most efficient AD-effluent to be added. In terms of composting process performance, the thermophilic phase of the amended reactor (A1) lasted 16 days and reached higher temperatures of about 72 °C, while the unamended one (A1) was characterized by a thermophilic temperature of around 66 °C indicating that the end products were of a pathogen-free compost. When it comes to the physico-chemical factors examined demonstrating that the biological conditions were sufficiently developed. The findings showed overall decreasing profiles during the composting period for moisture, C:N ratio as well as nitrification index (NI). From the quality-point of view, it was found that heavy metal concentrations had lower limits than those values set by German standards. Moreover, all the compost samples appeared to be stable and classified as class IV and V end product.
Adsorption into biochar-derived materials and mycoremediation are promising technologies for removing dyes from solid and liquid matrices. This study presents a combined treatment with adsorption into wood-chip biochar and mycodegradation under solid-state fermentation by Trametes villosa for removing the leather-dye Acid Blue 161. In the first stage, untreated wood-chip biochar, NaOH–depolymerised biochar and KMnO4–depolymerised biochar were assessed for their dye removal efficiency by adsorption. KMnO4–depolymerised biochar exhibited the highest adsorption (85.1 ± 1.9%) after 24 h of contact. KMnO4–depolymerisation modified some physical and chemical properties on the untreated wood-chip biochar, increasing the surface area (50.4 m2 g–1), pore size (1.9 nm), and presence of surface functional groups. Response surface methodology coupled with a Box–Behnken design was used to optimise the AB161 adsorption into the KMnO4–depolymerised biochar. The optimised conditions, pH 3.0, dye concentration 100 mg L–1 and sorbent dosage 2 g L–1, led to a higher dye removal efficiency by adsorption (91.9 ± 1.0%). In a second stage, the wood-chip biochar supplemented with nutrients (1% malt extract and 0.5% peptone) was employed as a solid matrix for growing T. villosa and regenerating the dye-saturated material. After 15 days, T. villosa was able to grow (86.8 ± 0.8%), exhibit laccase activity (621.9 ± 62.3 U L–1), and biodegrade (91.4 ± 1.3%) the dye adsorbed into the KMnO4–depolymerised biochar. Finally, the mycoregenerated biochar was reutilised in a new cycle of adsorption reaching 79.5 ± 2.0% of dye removal efficiency by adsorption. This study revealed the potential of the combined treatment and is an initial assessment for developing commercial alternatives for treating leather industry wastewaters.
Globally, the provision of energy is becoming an absolute necessity. Biomass resources are abundant and have been described as a potential alternative source of energy. However, it is important to assess the fuel characteristics of the various available biomass sources. Soft computing techniques are presented in this study to predict the mass yield (MY), energy yield (EY), and higher heating value (HHV) of hydrothermally carbonized biomass using Gene Expression Programming (GEP), multiple-input single output-artificial neural network (MISO-ANN), and Multilinear regression (MLR). The three techniques were compared using statistical performance metrics. The coefficient of determination (R 2), mean absolute error (MAE) and mean bias error (MBE) were used to evaluate the performance of the models. The MISO-ANN with 5-10 to 10-1 and 5-15-15-1 network architectures provided the most satisfactory performance of the three proposed models (R 2 = 0.976, 0.955, 0.996; MAE = 2.24, 2.11, 0.93; MBE = 0.16, 0.37, 0.12) for MY, EY and HHV, respectively. The GEP technique’s ability to predict hydrochar properties based on the input parameters was found to be satisfactory, while MLR provided an unsatisfactory predictive model. Sensitivity analysis was conducted, and the analysis revealed that volatile matter (VM) and temperature (Temp) have more influence on the MY, EY, and HHV.
The use of H9N2 subtype avian influenza vaccines is an effective approach for the control of the virus spread among the poultry, and for the upgrading of vaccine manufacturing, cell culture-based production platform could overcome the limitations of conventional egg-based platform and alternate it. The development of serum-free suspension cell culture could allow even higher virus productivity, where a suspension cell line with good performance and proper culture strategies are required. In this work, an adherent Mardin–Darby canine kidney (MDCK) cell line was adapted to suspension growth to cell concentration up to 12 × 106 cells/mL in a serum-free medium in batch cultures. Subsequently, the H9N2 influenza virus propagation in this MDCK cell line was evaluated with the optimization of infection conditions in terms of MOI and cell concentration for infection. Furthermore, various feed strategies were tested in the infection phase for improved virus titer and a maximum hemagglutinin titer of 13 log2 (HAU/50 μL) was obtained using the 1:2 medium dilution strategy. The evaluation of MDCK cell growth and H9N2 virus production in bioreactors with optimized operating conditions showed comparable cell performance and virus yield compared to shake flasks, with a high cell-specific virus yield above 13,000 virions/cell. With the purified H9N2 virus harvested from the bioreactors, the MDCK cell-derived vaccine was able to induce high titers of neutralizing antibodies in chickens. Overall, the results demonstrate the promising application of the highly efficient MDCK cell-based production platform for the avian influenza vaccine manufacturing.
The decomposition of lignocellulose in nature, as well as when used as feedstock in industrial settings, takes place in a dynamic system of biotic and abiotic reactions. In the present study, the impact of abiotic reactions during the storage of pretreated lignocellulose on the efficiency of subsequent saccharification was investigated. Abiotic decarboxylation was higher in steam-pretreated wheat straw (SWS, up till 1.5% CO2) than in dilute-acid-catalysed steam-pretreated forestry residue (SFR, up till 3.2% CO2) which could be due to higher iron content in SFR and there was no significant CO2 production in warm-water-washed slurries. Unwashed slurries rapidly consumed O2 during incubation at 50 °C; the behaviour was more dependent on storage conditions in case of SWS than SFR slurries. There was a pH drop in the slurries which did not correlate with acetic acid release. Storage of SWS under aerobic conditions led to oxidation of the substrate and reduced the extent of enzymatic saccharification by Cellic® CTec3. Catalase had no effect on the fractional conversion of the aerobically stored substrate, suggesting that the lower fractional conversion was due to reduced activity of the lytic polysaccharide monooxygenase component during saccharification. The fractional conversion of SFR was low in all cases, and cellulose hydrolysis ceased before the first sampling point. This was possibly due to excessive pretreatment of the forest residues. The conditions at which pretreated lignocellulose are stored after pretreatment significantly influenced the extent and kind of abiotic reactions that take place during storage. This in turn influenced the efficiency of subsequent saccharification. Pretreated substrates for laboratory testing must, therefore, be stored in a manner that minimizes abiotic oxidation to ensure that the properties of the substrate resemble those in an industrial setting, where pretreated lignocellulose is fed almost directly into the saccharification vessel.
