Combined alkali-photocatalytic stimulation enables click microbial domestication for boosted ammonia nitrogen removal.

Microbe-driven ammonia nitrogen removal plays a crucial role in the nitrogen cycle and wastewater treatment. However, the rational methods and mechanisms for boosting nitrogen conversion through microbial domestication are still limited. Herein, a combined alkali-photocatalytic stimulation strategy was developed to activate the Halomonas shizuishanensis DWK9 for efficient ammonia nitrogen removal. The strain DWK9 selected from saline-alkaline soil in Northwestern China possessed strong resistance to stress of saline-alkaline environment and free radicals, and was abundant in nitrogen conversion genes, thus is an ideal model for advanced microbial domestication. Bacterial in the combined alkali-photocatalytic stimulation group achieved a high ammonia nitrogen conversion rate of 67.5 %, 10 times outperforming the non-stimulated and single alkali/photocatalytic stimulation control groups. Morphology analysis revealed that the bacteria in the alkali-photocatalytic stimulated group formed a favorable structure for bioelectric transfer. Remarkably, the domesticated bacteria demonstrated improved electrochemical properties, including increased current capacity and lower overpotentials and impedance. Prokaryotic transcription genetic analysis together with qPCR analysis showed upregulation of denitrification-related metabolic pathway genes. A novel FAD dependent and NAD(P)H independent energy mode has been proposed. The universality and effectiveness of the as-developed combined alkali-photocatalytic microbial domestication strategy were further validated through indicator fish survival experiments. This work provides unprecedented degrees of freedom for the exploration of rational microbial engineering for optimized and controllable biogeochemical conversion.

Effects of Fishery Utilization on the Physicochemical Index and Microbial Community Composition in Saline-Alkaline Water.

Fishery utilization of idle saline-alkaline water resources offers various benefits including reducing surrounding soil salinity, improving the ecological environment, increasing arable land area, and providing economic advantages to the fishery industry. However, for decades, the characteristics and regulatory mechanisms of microbial communities that affect fishery utilization have not been clear, which restricts their application. In this study, high-throughput 16S rRNA amplicon sequencing was employed to analyze the bacterial community in these water resources. The sequencing yielded high-quality sequences (2,765,063), resulting in the identification of 18,761 bacterial operational taxonomic units (OTUs). Analysis revealed that the type of saline-alkaline water had a more significant influence on the bacterial community compared to seasonal variations within the aquaculture period. The Chao index for saline-alkaline ponds (ASW) was significantly lower (P < 0.05) than for still saline-alkaline water (SSW) and flowing saline-alkaline water (FSW), while the Shannon index for ASW was also significantly lower (P < 0.05) compared to FSW. When comparing ASW to nonaquaculture saline-alkaline water, a decrease in Proteobacteria to 26.87% was noted, particularly α-proteobacteria and γ-proteobacteria, accompanied by a rapid increase in Actinobacteria and Cyanobacteria to 28.60%. Networkx analysis further revealed that ASW significantly increased competition and amensalism from secondary saline-alkaline water microorganisms, resulting in a more solitary bacterial community composition as an adaptive strategy to cope with intense environmental pressures. Key bacterial species such as Pseudomonas, Hydrogenophaga, and Flavobacterium were found to be involved in hydrogen-cycling, nitrogen-cycling, and carbon-cycling, respectively, with all three exhibiting high abundance in FSW. Consequently, FSW demonstrates significant advantages in biogeochemical cycling, pollutant degradation, and the utilization of indigenous probiotic bacteria. Although the surface of abandoned secondary saline-alkaline land was covered with white salt particles, the fishery utilization of saline-alkaline water with low salinity levels (4.0-5.5), and the presence of nitrate and phosphate were identified as primary determinants of bacterial community composition. Nevertheless, a comparison of coastal high-salinity ponds indicated that salinity still selectively affects bacterial communities to some extent. Overall, our study provides valuable insights into the microbial regulation of nitrite during saline-alkaline water aquaculture, thereby aiding in the efficient utilization of secondary saline-alkaline water resources for fisheries.

Boosted growth performance, immunity, antioxidant capacity and disease resistance of crucian carp (Carassius auratus) by single or in combination dietary Bacillus subtilis and xylo-oligosaccharides.

In this study, a total of 420 healthy crucian carp (9.77 ± 0.04 g) were randomly divided into CK, B·S, XOS and B·S + XOS group, and cultured for 8 weeks. Results showed that the dietary Bacillus subtilis (B. subtilis) and xylo-oligosaccharides (XOS) can significantly increased the final weight, weight gain, specific growth rate, feed efficiency, protein efficiency and survival rate of crucian carp. Dietary B. subtilis and XOS can significantly increased the activities of catalase, glutathione, superoxide dismutase and total antioxidant capacity, significantly decreased the contents of malondialdehyde, and significantly increased the activities of alkaline phosphatase, acid phosphatase, lysozyme and the contents of complement component 3,4 and immunoglobulin M in crucian carp serum. In addition, compared with CK group, the expression levels of TGF-ß and IL-10 in B·S, XOS and B·S + XOS group were significantly increased, and the expression levels of TNF-α, HSP90, IL-1ß, TLR4 and MyD88 were significantly decreased. Supplementation of B. subtilis and XOS can also improve the intestinal tissue morphology of crucian carp. After injection of 1 × 107 CFU/mL Aeromonas hydrophila (A. hydrophila), compared with CK group, the survival rates of the B·S group, the XOS group and the B·S + XOS group were increased by 13.98%, 10.56% and 30.74%, respectively. These results show that dietary B. subtilis and XOS can significantly improve the growth performance, antioxidant capacity, immunity and resistance to A. hydrophila of crucian carp, and the combined effect is better than that of single addition.

Intestinal ion regulation exhibits a daily rhythm in Gymnocypris przewalskii exposed to high saline and alkaline water.

Naked carp (Gymnocypris przewalskii), endemic to the saline-alkaline Lake Qinghai, have the capacity to tolerate combinations of high salinity and alkalinity, but migrate to spawn in freshwater rivers each year. In this study, we measured the drinking rate over a 24 h period for naked carp exposed to saline-alkaline lake waters with salinities of 15 (L15) and 17 (L17). We also assessed the daily feed intakes of naked carp exposed to L15 and fresh water (FW). Additionally, we studied the daily expression of acid-base regulation and osmoregulation related genes and proteins in the intestine of naked carp exposed to saline-alkaline lake waters. Our results revealed that the drinking rate at night was significantly higher than in daytime when exposed to either L15 or L17, while feed intakes in daytime were significantly higher than at night. The relative expression of Na+/K+-ATPase α (NKA-α), solute carrier family members 26A6 (SLC26A6) and 4A4 (SLC4A4) in the intestine of naked carp exposed to L17 at night was higher than in daytime. Specifically, NKA-α mRNA expression at 4:00 was 7.22-fold and 5.63-fold higher than that at 10:00 and 16:00, respectively, and the expression at 22:00 was 11.29-fold and 8.80-fold higher than that at 10:00 and 16:00, respectively. Similarly, SLC26A6 mRNA expression was greatest at 22:00, exceeding that observed at 4:00, 10:00 and 16:00 by 3.59, 4.44 and 11.14-fold, respectively. Finally, the expression of NKA-α and SLC26A6 protein at the single cell level was also higher at night than during the day, which was 1.65-fold and 1.37-fold higher at 22:00 respectively compared to 16:00. Overall, the present findings revealed that naked carp drinks at night and feeds during the day, demonstrating that intestinal ion regulation exhibits a daily rhythm when exposed to high saline and alkaline lake water.

Ammonia excretion and blood gas variation in naked carp (Gymnocypris przewalskii) exposed to acute hypoxia and high alkalinity.

Naked carp (Gymnocypris przewalskii), endemic to the saline-alkaline Lake Qinghai, have the capacity to tolerate combined hypoxia and high alkalinity. This study evaluated the effect of the interaction between carbonate alkalinity and hypoxia on ammonia excretion and blood gas variation in naked carp. Naked carp were subjected to normoxic, hypoxic and reoxygenation phases at two different carbonate alkalinity levels (CA0 = 0 mmol/L; CA32 = 32 mmol/L) for 4 days. The ammonia excretion rate (JAmm) of the CA0 group rapidly decreased under hypoxia and recovered under normoxia for four consecutive days. The JAmm under CA32 also decreased under hypoxia and recovered to its previous level in the first 2 days. However, the JAmm under CA32 was lower than that under CA0. The blood pO2, sO2 of CA0 and CA32 group was significantly reduced under hypoxia, after which both groups recovered. Blood pCO2 of the CA32 group was lower than CA0 throughout the experiment. There were no changes in haematocrit of the naked carp exposed to carbonate alkalinity and hypoxia. The alkaline water increased the pH of the blood and contributed to increased haemoglobin O2 affinity. Overall, the present findings reveal that naked carp is a tolerant species that can maintain main ionic homeostasis under severe alkalinity and hypoxia. The high alkaline water is beneficial for naked carp to adapt to hypoxic environment.

Acute Exposure to key Aquaculture Environmental Stressors Impaired the Aerobic Metabolism of Carassius auratus gibelio.

Carassius auratus gibelio is an omnivore favored for its flavor and is commonly used as a benthic species in traditional pond polyculture. This study investigated the effects of common aquaculture stressors, such as high ammonia, high nitrite, high pH, and hypoxia on the aerobic metabolism of C. auratus gibelio. The results showed that the standard metabolic rate (SMR) was positively correlated with ammonia, nitrite, and pH, while the maximum metabolic rate (MMR) was negatively correlated with all four stressors. Thus, aerobic scope (AS) was reduced when C. auratus gibelio was exposed to high ammonia, high nitrite, high pH, and hypoxia. The peak of post-prandial O2 consumption was positively correlated with nitrite, pH, and the occurrence of the peak metabolic rate post-prandial was delayed in high ammonia, high nitrite, hypoxia, and high pH conditions. These findings indicated that, in experimental conditions, exposure to these environmental stressors can influence aerobic metabolism in C. auratus gibelio. With more energy required to maintain standard metabolic rates, less will be available for growth. While the C. auratus gibelio is one of the most hypoxia tolerance species, the reduction we observed in AS caused by stressors that commonly occur in ponds and in nature will likely affect growth in ponds and fitness in nature. These data have provided insight into the optimal, fitness-maximizing thresholds for these common stressors in this species of interest.

Gymnocypris przewalskii decreases cytosolic carbonic anhydrase expression to compensate for respiratory alkalosis and osmoregulation in the saline-alkaline lake Qinghai.

Naked carp (Gymnocypris przewalskii), endemic to the saline-alkaline Lake Qinghai, have the capacity to tolerate combined high salinity and alkalinity, but migrate to spawn in freshwater rivers each year. In this study, the full-length cDNA of the cytosolic carbonic anhydrase c isoform of G. przewalskii (GpCAc) was amplified and sequenced; mRNA levels and enzyme activity of GpCAc and blood chemistry were evaluated to understand the compensatory responses as the naked carp returned to the saline-alkaline lake after spawning. We found that GpCAc had a total length of 1400 bp and encodes a peptide of 260 amino acids. Comparison of the deduced amino acid sequences and phylogenetic analysis showed that GpCAc was a member of the cytosolic carbonic anhydrase II-like c family. Cytosolic-carbonic-anhydrase-c-specific primers were used to analyze the tissue distribution of GpCAc mRNA expression. Expression of GpCAc mRNA was found in brain, gill, liver, kidney, gut, and muscle tissues, but primarily in the gill and posterior kidney; however, none was evident in red blood cells. Transferring fish from river water to lake water resulted in a respiratory alkalosis, osmolality, and ion rise in the blood, as well as significant decreases in the expression and enzyme activity of GpCAc in both the gill and kidney within 96 h. These results indicate that GpCAc may play an important role in the acclimation to both high salinity and carbonate alkalinity. Specifically, G. przewalskii decreases cytosolic carbonic anhydrase c expression to compensate for a respiratory alkalosis and to aid in osmoregulation during the transition from river to saline-alkaline lake.

Carbonic anhydrase 2-like and Na⁺-K⁺-ATPase α gene expression in medaka (Oryzias latipes) under carbonate alkalinity stress.

High carbonate alkalinity is one of the major stress factors for living organisms in saline-alkaline water areas. Acute and chronic effects of carbonate alkalinity on expression of two genes, carbonic anhydrase 2-like (CA2-like) and Na(+)-K(+)-ATPase α subunit (NKA-α) mRNA in medaka (Oryzias latipes) were evaluated to better understand the responses important for coping with a carbonate alkalinity stress. In the acute exposure experiment, the expression of CA2-like and NKA-α mRNA in the gill and kidney of medaka were examined from 0 h to 7 days exposed to 30.4 mM carbonate alkalinity water. Exposure to high carbonate alkalinity resulted in a transitory alkalosis, followed by a transient increase in gill and kidney CA2-like and NKA-α mRNA expression. In the chronic exposure experiment, the expression of these two genes was examined in the gill and kidney at 50 days post-exposure to six different carbonate alkalinity concentrations ranging from 1.5 to 30.4 mM. Gill and kidney CA2-like mRNA levels in 30.4 mM were approximately 10 and 30 times higher than that of the control (1.5 mM), respectively. Less differences were found in NKA-α expression in the 50-days exposure. The results indicate that when transferred to high carbonate alkalinity water, a transitory alkalosis may occur in medaka, followed by compensatory acid-base and ion regulatory responses. Thus, CA2-like and NKA-α are at least two of the important factors that contribute to the regulation of alkalinity stress.

A modified method to detect the phagocytic ability of eosinophilic and basophilic haemocytes in the oyster Crassostrea plicatula.

The immune defence system of bivalve species largely depends on haemocytes. Haemocytes are generally classified as hyalinocytes (H) or granulocytes (G), and each cell type is further sub-classified as eosinophilic (E) or basophilic (B) haemocytes. Until recently, research on eosinophilic and basophilic haemocytes has primarily focused on their morphologies, dye affinities and intracellular components. Few studies have investigated their phagocytic ability because of the absence of appropriate experimental methods. In this study, we introduce a modified method suitable to detect the phagocytic ability of eosinophilic and basophilic haemocytes. This modified method involves neutral red staining by employing fluorescent microspheres as the phagocytosed medium. Specifically, haemocytes are incubated with fluorescent microspheres and then stained with neutral red. Next, the stained haemocytes are fixed by acetone and are counterstained by propidium iodide. Finally, the haemocytes are observed under a multifunctional microscope to analyse the phagocytic ability by counting the number of eosinophilic or basophilic haemocytes involved in phagocytosis (calculation for phagocytic rate, PR) and the number of phagocytosed microspheres by each eosinophilic or basophilic haemocyte (calculation for phagocytic index, PI). By employing this modified method in the oyster Crassostrea plicatula, we found that the PRs of G and H were very similar to the data obtained by another method, flow cytometry, indicating that this modified method has high accuracy. Additionally, we also found that the PR and PI in E-G were 70.9 ± 7.3% and 1.0 ± 0.2, respectively, which were both significantly higher than those in B-G (53.1 ± 6.4% and 0.7 ± 0.1). The PR and PI in E-H were 16.3 ± 2.8% and 0.2 ± 0.1, respectively, while in B-H, the PR and PI were 13.3 ± 3.6% and 0.2 ± 0.1, respectively, with no significant difference observed. Based on this result, eosinophilic granulocytes are more active in phagocytosis than basophilic granulocytes in the oyster immune defence system.

Combined effects of carbonate alkalinity and pH on survival, growth and haemocyte parameters of the Venus clam Cyclina sinensis.

Carbonate alkalinity (CA) and pH are considered to be two important stress factors that determine the response of aquatic animals to sudden transfers into saline-alkaline water. To evaluate the potential for aquaculture production of Venus clams (Cyclina sinensis) farmed in saline-alkaline water, the combined effects of CA (2.5 (control), 10.0, 20.0 and 40.0 meq/l) and pH (8.0 (control), 8.5, 9.0 and 9.5) on survival rate was monitored every day for 10 days. Length gain rate (LGR) and weight gain rate (WGR) were also monitored for two months, and total haemocyte count (THC), phagocytic rate (PR) and haemocyte mortality (HM) were measured for 3, 6, 12 and 24 days under the same water temperature (20 °C) and salinity (15‰) conditions. The results showed that survival rates in treatments of CA ≤ 20.0, combined with pH ≤ 9.0, were 100%. LGR and WGR in treatments of CA 2.5 & pH 8.0 (control), CA 2.5 & pH 8.5 and CA 10.0 & pH 8.0 exhibited the largest values (P > 0.05), while in other treatments, they showed a decreasing trend with an increase in either CA or pH or both (P < 0.05). Similarly, for THC, PR and HM, no significant differences were observed among the fast growth treatments during the entire experimental period (P > 0.05), however, in other treatments, they presented significant differences, especially on day 3 and 6 (P < 0.05), most notably with increases in CA or pH, but returned to control levels on day 12. In conclusion, in this study, a strong interaction between CA and pH was observed. Additionally, it was ascertained that the Venus clam C. sinensis can withstand the stress of CA 20.0 combined pH 9.0, although individuals grows slowly and may take approximately 12 days to recover to the unstressed condition.