Biodegradable chelating agents for enhancing phytoremediation: Mechanisms, market feasibility, and future studies.

Heavy metals in soil significantly threaten human health, and their remediation is essential. Among the various techniques used, phytoremediation is one of the safest, most innovative, and effective. In recent years, the use of biodegradable chelators to assist plants in improving their remediation efficiency has gained popularity. These biodegradable chelators aid in the transformation of metal ions or metalloids, thereby facilitating their mobilization and uptake by plants. Developed countries are increasingly adopting biodegradable chelators for phytoremediation, with a growing emphasis on green manufacturing and technological innovation in the chelating agent market. Therefore, it is crucial to gain a comprehensive understanding of the mechanisms and market prospects of biodegradable chelators for phytoremediation. This review focuses on elucidating the uptake, translocation, and detoxification mechanisms of chelators in plants. In this study, we focused on the effects of biodegradable chelators on the growth and environmental development of plants treated with phytoremediation agents. Finally, the potential risks associated with biodegradable chelator-assisted phytoremediation are presented in terms of their availability and application prospects in the market. This study provides a valuable reference for future research in this field.

Development of a Strategy for L-Lactic Acid Production by Rhizopus oryzae Using Zizania latifolia Waste and Cane Molasses as Carbon Sources.

As a biodegradable and renewable material, polylactic acid is considered a major environmentally friendly alternative to petrochemical plastics. Microbial fermentation is the traditional method for lactic acid production, but it is still too expensive to compete with the petrochemical industry. Agro-industrial wastes are generated from the food and agricultural industries and agricultural practices. The utilization of agro-industrial wastes is an important way to reduce costs, save energy and achieve sustainable development. The present study aimed to develop a method for the valorization of Zizania latifolia waste and cane molasses as carbon sources for L-lactic acid fermentation using Rhizopus oryzae LA-UN-1. The results showed that xylose derived from the acid hydrolysis of Z. latifolia waste was beneficial for cell growth, while glucose from the acid hydrolysis of Z. latifolia waste and mixed sugars (glucose and fructose) from the acid hydrolysis of cane molasses were suitable for the accumulation of lactic acid. Thus, a three-stage carbon source utilization strategy was developed, which markedly improved lactic acid production and productivity, respectively reaching 129.47 g/L and 1.51 g/L·h after 86 h of fermentation. This work demonstrates that inexpensive Z. latifolia waste and cane molasses can be suitable carbon sources for lactic acid production, offering an efficient utilization strategy for agro-industrial wastes.

Isolation and screening of a chitin deacetylase producing Bacillus cereus and its potential for chitosan preparation.

Chitosan is a biopolymer material extracted from marine biomass waste such as shrimp and crab shells, which has good biocompatibility and degradability with great potential for application in the field of wastewater treatment and soil remediation. The higher the degree of deacetylation (DD), the better the adsorption performance of chitosan. Chitin deacetylase (CDA) can specifically catalyze the deacetylate of chitin in a green reaction that is environmentally friendly. However, the scarcity of high yielding chitin deacetylase strains has been regarded as the technical bottleneck of chitosan green production. Here, we screened a natural chitin degrading bacterium from coastal mud and identified it as Bacillus cereus ZWT-08 by re-screening the chitin deacetylase activity and degree of deacetylation values. By optimizing the medium conditions and enzyme production process, ZWT-08 was cultured in fermentation medium with 1% (m/V) glucose and yeast extract at pH 6.0, 37°C, and a stirring speed of 180 r/min. After fermenting in 5 L fermenter for 48 h, the deacetylation activity of the supernatant reached 613.25 U/mL. Electron microscopic examination of the chitin substrate in the fermentation medium revealed a marshmallow-like fluffy texture on its structural surface. Meanwhile, 89.29% of the acetyl groups in this chitin substrate were removed by enzymatic digestion of chitin deacetylase produced by ZWT-08, resulting in the preparation of chitosan a degree of deacetylation higher than 90%. As an effective strain for chitosan production, Bacillus cereus ZWT-08 plays a positive role in the bioconversion of chitin and the upgrading of the chitosan industry.

Efficient Co-production of Docosahexaenoic Acid Oil and Carotenoids in Aurantiochytrium sp. Using a Light Intensity Gradient Strategy.

Aurantiochytrium is a promising source of docosahexaenoic acid (DHA) and carotenoids, but their synthesis is influenced by environmental stress factors. In this study, the effect of different light intensities on the fermentation of DHA oil and carotenoids using Aurantiochytrium sp. TZ209 was investigated. The results showed that dark culture and low light intensity conditions did not affect the normal growth of cells, but were not conducive to the accumulation of carotenoids. High light intensity promoted the synthesis of DHA and carotenoids, but caused cell damage, resulting in a decrease of oil yield. To solve this issue, a light intensity gradient strategy was developed, which markedly improved the DHA and carotenoid content without reducing the oil yield. This strategy produced 30.16 g/L of microalgal oil with 15.11 g/L DHA, 221 µg/g astaxanthin, and 386 µg/g ß-carotene. This work demonstrates that strain TZ209 is a promising DHA producer and provides an efficient strategy for the co-production of DHA oil together with carotenoids.

Simultaneous improvement of the thermostability and activity of lactic dehydrogenase from Lactobacillus rossiae through rational design.

d-Phenyllactic acid, is a versatile organic acid with wide application prospects in the food, pharmaceutical and material industries. Wild-type lactate dehydrogenase LrLDH from Lactobacillus rossiae exhibits a high catalytic performance in the production of d-phenyllactic acid from phenylpyruvic acid or sodium phenylpyruvate, but its industrial application is hampered by poor thermostability. Here, computer aided rational design was applied to improve the thermostability of LrLDH. By using HotSpot Wizard 3.0, five hotspot residues (N218, L237, T247, D249 and S301) were identified, after which site-saturation mutagenesis and combined mutagenesis were performed. The double mutant D249A/T247I was screen out as the best variant, with optimum temperature, t 1/2, and T 10 50 that were 12 °C, 17.96 min and 19 °C higher than that of wild-type LrLDH, respectively. At the same time, the k cat/K m of D249A/T247I was 1.47 s-1 mM-1, which was 3.4 times higher than that of the wild-type enzyme. Thus rational design was successfully applied to simultaneously improve the thermostability and catalytic activity of LrLDH to a significant extent. The results of molecular dynamics simulations and molecular structure analysis could explain the mechanisms for the improved performance of the double mutant. This study shows that computer-aided rational design can greatly improve the thermostability of d-lactate dehydrogenase, offering a reference for the modification of other enzymes.

Screening of highly effective mixed natural antioxidants to improve the oxidative stability of microalgal DHA-rich oil.

Docosahexaenoic acid (DHA)-rich oil sourced from microalgae can easily become oxidized. The objective of this work was to screen the optimal natural antioxidant mixture for protecting DHA-rich oil. Different natural antioxidants, encompassing tea polyphenols, natural vitamin E, rosemary extract, licorice root antioxidant, ascorbyl palmitate and lecithin were tested individually and in combination in an accelerated oxidation process. Three antioxidants namely natural vitamin E, rosemary extract and ascorbyl palmitate with synergistic effects were chosen, and their concentrations were further optimized using response-surface methodology. The highest antioxidants activity of 16.1740 was obtained with a combination of 0.0224% vitamin E, 0.0259% rosemary extract and 0.0166% ascorbyl palmitate, which prolonged the time until oxidation induction to 20.21 days. The mixed natural antioxidants showed a similar antioxidant effect to 0.02% tert-butylhydroquinone and was better than 0.02% butylated hydroxyanisole. These data indicate that the mixed natural antioxidants optimized in this work can be directly applied in the protection of commercial microalgal DHA-rich oil.

Effects of regulating miR-132 mediated GSK-3ß on learning and memory function in mice.

The aimf of this study was to explore effects of miR-132 and glycogen synthase kinase-3ß (GSK-3ß) on learning and memory in mice. miR-132 inhibitor GSK-3ß overexpression agent (sh-GSK-3ß) and normal saline (negative control group) were injected into the hippocampus of adult mice, and healthy adult mice were taken as the unrelated control group. The expression of miR-132 and GSK-3ß in the hippocampus of adult and elderly mice was detected using reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. Morris water maze test was employed to detect learning and memory function in mice. The dual luciferase reporter was adopted to determine the relationship between miR-132 and GSK-3ß. Compared with the adult group, the expression of miR-132 was significantly downregulated in the hippocampus in the elderly group, while the expression of GSK-3ß was upregulated. Injecting miR-132 inhibitor into the hippocampus of adult mice led to a significant increase in escape latency and a significant decrease in the number of times of crossing platforms. The injection of GSK-3ß overexpression agent into the hippocampus of adult mice resulted in a marked increase in escape latency and a significant decrease in the number of times of crossing platforms in the water maze test. It was also found that downregulation of GSK-3ß reversed the decline in learning and memory in mice caused by downregulation of miR-132 expression. The dual luciferase report identified a targeted regulatory relationship between miR-132 and GSK-3ß. Overexpression of miR-132 can inhibit the expression of GSK-3ß in mouse learning and memory ability, which provides some inspiration for understanding the occurrence of learning and memory disorders and future treatment methods.

Comparative proteomic analysis of Rhizopus oryzae hyphae displaying filamentous and pellet morphology.

Industrial strains of Rhizopus oryzae is known for its strong ability to produce L-( +)-lactic acid, ethanol, and fumaric acid at high yields. To better understand the underlying mechanism behind the physiology of R. oryzae, we conducted the proteome changes between two different morphologies using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. R. oryzae exhibited pellet morphology and filamentous morphology when the initial pH of the culture medium was 3.0 and 5.0, respectively. The concentration of lactic acid reached 63.5 g L-1 in the samples containing the pellet morphology, compared to 41.5 g L-1 produced by filamentous R. oryzae. Proteomic analysis indicated that expression levels of 128 proteins changed significantly. Of these, 17 protein spots were successfully identified by mass spectrometry and were deemed to be mainly involved in carbohydrate metabolism, genetic information processing, chitin metabolism, protein catabolism, protein folding, and antioxidative pathway. L-lactate dehydrogenase (RO3G_06188), enolase (RO3G_05466) and 2, 3-bisphosphoglycerate-independent phosphoglycerate mutase (RO3G_02462) were found to be upregulated, while isocitrate dehydrogenase (RO3G_13820) was downregulated in the samples with pellet morphology compared to the filamentous hyphae. These results suggested that more carbon flow was directed towards lactic acid biosynthesis in R. oryzae hyphae with pellet morphology.

Enzymological characterization of a novel d-lactate dehydrogenase from Lactobacillus rossiae and its application in d-phenyllactic acid synthesis.

A novel lactate dehydrogenase gene, named lrldh, was cloned from Lactobacillus rossiae and heterologously expressed in Escherichia coli. The lactate dehydrogenase LrLDH is NADH-dependent with a molecular weight of approximately 39 kDa. It is active at 40 °C and pH 6.5 and stable in a neutral to alkaline environment below 35 °C. The kinetic constants, including maximal reaction rate (V max), apparent Michaelis-Menten constant (K m), turnover number (K cat) and catalytic efficiency (K cat/K m) for phenylpyruvic acid were 1.95 U mg-1, 2.83 mM, 12.29 s-1, and 4.34 mM-1 s-1, respectively. Using whole cells of recombinant E. coli/pET28a-lrldh, without coexpression of a cofactor regeneration system, 20.5 g l-1 d-phenyllactic acid with ee above 99% was produced from phenylpyruvic acid in a fed-batch biotransformation process, with a productivity of 49.2 g l-1 d-1. Moreover, LrLDH has broad substrate specificity to a range of ketones, keto acids and ketonic esters. Taken together, LrLDH is a promising biocatalyst for the efficient synthesis of d-phenyllactic acid and other fine chemicals.

Development of a strategy for the production of docosahexaenoic acid by Schizochytrium sp. from cane molasses and algae-residue.

The aim of this work was to reduce the algae-residue emission and make use of cane molasses as fermentation materials for docosahexaenoic acid (DHA) fermentaion by Schizochytrium sp., which further could cut the cost of DHA production. Algae-residue and cane molasses were respectively used as nitrogen and carbon sources to replace yeast extract and glucose. A significant DHA yield of 18.58 g/L was obtained using algae-residue, while cane molasses could not be used well as sole carbon source due to the presence of undesirable substance. A two-stage culture strategy with glucose followed by pretreated cane molasses as carbon source was developed, resulting in a final DHA yield of 15.22 g/L. This study therefore offers an economical and green strategy for DHA production by Schizochytrium sp.

Development of a multi-stage continuous fermentation strategy for docosahexaenoic acid production by Schizochytrium sp.

Docosahexaenoic acid (DHA) has wide-ranging benefits for normal development of the visual and nervous systems in infants. A sustainable source of DHA production through fermentation using Schizochytrium sp. has been developed. In this paper, we present the discovery of growth-uncoupled DHA production by Schizochytrium sp. and the development of corresponding kinetic models of fed-batch fermentations, which can be used to describe and predict the cell growth and substrate utilization as well as lipid and DHA production. Based on this kinetic model, a predictive model of multi-stage continuous fermentation process was established and used to analyze, optimize and design the process parameters. Optimal predicted processes of two-stage and three-stage continuous fermentation were developed and verified in lab-scale bioreactor based on the predicted process parameters. A successful three-stage continuous fermentation was achieved, which increased the lipid, DHA content and DHA productivity by 47.6, 64.3 and 97.1%, respectively, compared with two-stage continuous fermentation.

Development of a method for the valorization of fermentation wastewater and algal-residue extract in docosahexaenoic acid production by Schizochytrium sp.

Fermentation wastewater (FW) and algal residue are major by-products of docosahexaenoic acid (DHA) fermentations utilizing Schizochytrium sp. In order to reduce production costs and environmental pollution, we explored the application of FW and algal-residue extract (AE) for DHA production. Components analysis showed that FW and AE contained some mineral elements and protein residues, respectively. When they were used for DHA fermentation, results showed that 20% replacement of fresh water by FW and 80% replacement of yeast extract nitrogen by AE reached DHA content of 22.23 g/L and 27.10 g/L, respectively. Furthermore, a novel medium that utilizes a mixture of FW and AE was applied for DHA fermentation, whereby the final DHA yield reached 28.45 g/L, 24.56% higher than conventional medium. The strategy of valorizing fermentation waste provides a new method for reducing the costs and reducing environmental pollution of microbial fermentations.

Development of a real-time bioprocess monitoring method for docosahexaenoic acid production by Schizochytrium sp.

Oxygen uptake rate (OUR) and respiratory quotient (RQ) are key respiratory parameters for docosahexaenoic acid (DHA) production by Schizochytrium sp. HX-308 under dissolved oxygen limited conditions. To investigate the relationship of OUR and RQ with culture status, three independent cultures with different aeration rates were performed in a 50L bioreactor. OUR was found to be positively correlated with the aeration rate, which reflected the oxygen supply level in each culture. The highest biomass, reaching 124.5g/L, was achieved under the highest OUR. DHA content was found to be highly correlated with the RQ value, and the highest DHA content (44.85% in total fatty acids, w/w) was achieved in the highest RQ level, which implies that the polyketide synthase pathway was more active. OUR and RQ, which reflect the physiological state of microorganisms, are suggested as synergistic real-time bioprocess monitoring parameters for DHA fermentation.

Effect of CO2 supply conditions on lipid production of Chlorella vulgaris from enzymatic hydrolysates of lipid-extracted microalgal biomass residues.

The hydrolysates from lipid-extracted microalgal biomass residues (LMBRs) were used as a source of nutrients for the cultivation of Chlorella vulgaris for lipid production under various CO(2) supply conditions, including different CO(2) concentrations and aeration rates. Both parameters had a significant effect on lipid production. A CO(2) concentration of 5% was found to be most suitable for microalgal growth. Microalga grew best at a CO(2) aeration rate of 0.5 vvm. At this rate, biomass concentration and lipid productivity were at a maximum of 3.83 g L(-1) and 157 mg L(-1)d(-1), respectively, but decreased at lower or higher aeration rates. The present results showed that LMBRs utilization was effective in microalgal lipid production under suitable CO(2) supply conditions.

Lipid production of Chlorella vulgaris from lipid-extracted microalgal biomass residues through two-step enzymatic hydrolysis.

Lipid-extracted microalgal biomass residues (LMBRs) were treated using cellulase, neutrase and alcalase in a two-step process and the resulting hydrolysates were used as a source of nutrients for the cultivation of Chlorella vulgaris under non-aerated and aerated conditions for lipid production. Aeration was favorable for cell growth and lipid accumulation and a biomass of approximately 3.28 g L(-1), lipid content of 35% and lipid productivity of 116 mg L(-1) d(-1) were obtained. Thus, the tested mode of LMBRs utilization was effective for nutrient recycling in microalgal biodiesel production.