Survey of the Prosthogonimus spp. metacercariae infection in the second intermediate host dragonfly in Heilongjiang Province, China.

Prosthogonimiasis poses a threat to the reproductive system of poultry and wild birds, which are the definitive hosts of the parasite causing this disease. However, the parasite infection of the second intermediate host (dragonfly), the primary vector of this pathogen, is rarely reported. In this study, the prevalence of Prosthogonimus infection in dragonflies was investigated from June 2019 to October 2022 in Heilongjiang Province, northeast China. The species of metacercariae isolated from dragonfly were identified by morphological characteristics, molecular biology techniques, and animal infection experiments. The results showed that 11 species of dragonflies and one damselfly were identified and among six of the dragonflies infected by Prosthogonimus metacercariae, Sympetrum depressiusculum (28.53%) had the highest infection rate among all positive dragonflies, followed by Sympetrum vulgatum (27.86%) and Sympetrum frequens (20.99%), which are preferred hosts, and the total prevalence was 20.39% (2061/10,110) in Heilongjiang Province. Three species of Prosthogoniumus metacercariae were isolated, including Prosthogonimus cuneatus, Prosthogonimus pullucidus, and Prosthogonimus sp., among which P. cuneatus was the dominant species in dragonflies in Heilongjiang Province. This is the first report on the prevalence of Prosthogonimus in dragonflies in China, which provides baseline data for the control of prosthogonimiasis in Heilongjiang Province and a reference for the prevention of prosthogonimiasis in other areas of China.

The complete mitochondrial genome of Ogmocotyle ailuri: gene content, composition and rearrangement and phylogenetic implications.

Trematodes of the genus Ogmocotyle are intestinal flukes that can infect a variety of definitive hosts, resulting in significant economic losses worldwide. However, there are few studies on molecular data of these trematodes. In this study, the mitochondrial (mt) genome of Ogmocotyle ailuri isolated from red panda (Ailurus fulgens) was determined and compared with those from Pronocephalata to investigate the mt genome content, genetic distance, gene rearrangements and phylogeny. The complete mt genome of O. ailuri is a typical closed circular molecule of 14 642 base pairs, comprising 12 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and 2 non-coding regions. All genes are transcribed in the same direction. In addition, 23 intergenic spacers and 2 locations with gene overlaps were determined. Sequence identities and sliding window analysis indicated that cox1 is the most conserved gene among 12 PCGs in O. ailuri mt genome. The sequenced mt genomes of the 48 Plagiorchiida trematodes showed 5 types of gene arrangement based on all mt genome genes, with the gene arrangement of O. ailuri being type I. Phylogenetic analysis using concatenated amino acid sequences of 12 PCGs revealed that O. ailuri was closer to Ogmocotyle sikae than to Notocotylus intestinalis. These data enhance the Ogmocotyle mt genome database and provide molecular resources for further studies of Pronocephalata taxonomy, population genetics and systematics.

A robust soft sensor based on artificial neural network for monitoring microbial lipid fermentation processes using Yarrowia lipolytica.

Microbial oils produced by Yarrowia lipolytica offer an environmentally friendly and sustainable alternative to petroleum as well as traditional lipids from animals and plants. The accurate measurement of fermentation parameters, including the substrate concentration, dry cell weight, and lipid accumulation, is the foundation of process control, which is indispensable for industrial lipid production. However, it remains a great challenge to measure the complex parameters online during the lipid fermentation process, which is nonlinear, multivariate, and characterized by strong coupling. As a type of AI technology, the artificial neural network model is a powerful tool for handling extremely complex problems, and it can be employed to develop a soft sensor to monitor the microbial lipid fermentation process of Y. lipolytica. In this study, we first analyzed and emphasized the volume of sodium hydroxide and dissolved oxygen concentration as central parameters of the fermentation process. Then, a soft sensor based on a four-input artificial neural network model was developed, in which the input variables were fermentation time, dissolved oxygen concentration, initial glucose concentration, and additional volume of sodium hydroxide. This provides the possibility of online monitoring of dry cell weight, glucose concentration, and lipid production with high accuracy, which can be extended to similar fermentation processes characterized by the addition of bases or acids, as well as changes of the dissolved oxygen concentration.

Characterization of the complete mitochondrial genomes of Diplodiscus japonicus and Diplodiscus mehari (Trematoda: Diplodiscidae): Comparison with the members of the superfamily Paramphistomoidea and phylogenetic implication.

Diplodiscus japonicus and Diplodiscus mehari (Trematoda: Diplodiscidae) are two important parasites in wood frogs, which have large infection rates and essential importance of ecology, economy and society. In this study, the complete mitochondrial (mt) genomes of D. japonicus and D. mehari were sequenced, then compared with other related trematodes in the superfamily Paramphistomoidea. The complete circular mt sequence of D. japonicus and D. mehari were 14,210 bp and 14,179 bp in length, respectively. Both mt genomes comprised 36 functional subunits, consisting of 12 protein-coding genes (PCGs), two ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and one non-coding region. The mt genes of D. japonicus and D. mehari were transcribed in the same direction, and the gene arrangements were identical to those of Paramphistomoidea trematodes. In the 12 PCGs, GTG was the most common initiation codon, whereas TAG was the most common termination codon. All tRNAs had a typical cloverleaf structure except tRNA Ser1. A comparison with related Paramphistomoidea trematode mt genomes suggested that the cox1 gene of D. mehari was the longest in these trematodes. Phylogenetic analyses revealed that Paramphistomoidea trematodes formed a monophyletic branch, Paramphistomidae and Gastrothylacidae were more closely related than Diplodiscidae. And the further analysis with Pronocephalata branch found that the flukes parasitic in amphibians (frogs) formed one group, and the flukes from ruminants (cattle, sheep, ect) formed another group. Our study demonstrated the importance of sequencing mt genomes of D. japonicus and D. mehari, which will provide significant molecular resources for further studies of Paramphistomoidea taxonomy, population genetics and systematics.

Corrigendum: Key Enzymes in Fatty Acid Synthesis Pathway for Bioactive Lipids Biosynthesis.

[This corrects the article DOI: 10.3389/fnut.2022.851402.].

Key Enzymes in Fatty Acid Synthesis Pathway for Bioactive Lipids Biosynthesis.

Dietary bioactive lipids, one of the three primary nutrients, is not only essential for growth and provides nutrients and energy for life's activities but can also help to guard against disease, such as Alzheimer's and cardiovascular diseases, which further strengthen the immune system and maintain many body functions. Many microorganisms, such as yeast, algae, and marine fungi, have been widely developed for dietary bioactive lipids production. These biosynthetic processes were not limited by the climate and ground, which are also responsible for superiority of shorter periods and high conversion rate. However, the production process was also exposed to the challenges of low stability, concentration, and productivity, which was derived from the limited knowledge about the critical enzyme in the metabolic pathway. Fortunately, the development of enzymatic research methods provides powerful tools to understand the catalytic process, including site-specific mutagenesis, protein dynamic simulation, and metabolic engineering technology. Thus, we review the characteristics of critical desaturase and elongase involved in the fatty acids' synthesis metabolic pathway, which aims to not only provide extensive data for enzyme rational design and modification but also provides a more profound and comprehensive understanding of the dietary bioactive lipids' synthetic process.

Characterization of the mitochondrial genome of Tetrameres grusi and insights into the phylogeny of Spirurina.

Tetrameres grusi is a significant parasitic nematode of cranes that is classified into suborder Spirurina. However, for more than a century, this classification has been controversial. Mitochondrial genomes are valuable resources for parasite taxonomy, population genetics and systematics studies. Here, the mitochondrial genome of T. grusi was determined and subsequently compared with those from Spirurina species using concatenated datasets of amino acid sequences predicted from mitochondrial protein-coding genes. The complete mitochondrial genome of T. grusi is circular with 13,709 bp, and it contains 12 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one non-coding region. All of the protein-coding genes are transcribed in the same direction. There were 18 intergenic spacers of 1-44 bp, and six locations with gene overlaps, ranging from 1 bp to 28 bp, in the mitochondrial genome of T. grusi. The AT content of this mitochondrial genome was 71.56%. This was similar to mitochondrial genomes of other Spirurina species, which also exhibited strong AT content bias, not only in the nucleotide composition but also in codon usage. The sequenced mitogenomes of the 25 Spirurina nematodes showed three classes of gene arrangements based on the 12 protein-coding genes, and the gene arrangement of the T. grusi mitochondrial genome belonged to the Class I. Phylogenetic analyses using mitochondrial genomes of 25 Spirurina nematodes revealed that T. grusi (Habronematoidea) was closer to Gongylonema pulchrum (Spiruroidea) than Spirocerca lupi (Thelazioidea). The availability of the complete mitochondrial genome sequence of T. grusi provides new and useful genetic markers for further studies on Spirurina nematodes.

A robust soft sensor to monitor 1,3-propanediol fermentation process by Clostridium butyricum based on artificial neural network.

With the aggravation of environmental pollution and energy crisis, the sustainable microbial fermentation process of converting glycerol to 1,3-propanediol (1,3-PDO) has become an attractive alternative. However, the difficulty in the online measurement of glycerol and 1,3-PDO creates a barrier to the fermentation process and then leads to the residual glycerol and therefore, its wastage. Thus, in the present study, the four-input artificial neural network (ANN) model was developed successfully to predict the concentration of glycerol, 1,3-PDO, and biomass with high accuracy. Moreover, an ANN model combined with a kinetic model was also successfully developed to simulate the fed-batch fermentation process accurately. Hence, a soft sensor from the ANN model based on NaOH-related parameters has been successfully developed which cannot only be applied in software to solve the difficulty of glycerol and 1,3-PDO online measurement during the industrialization process, but also offer insight and reference for similar fermentation processes.

Metabolic profiles analysis of 1,3-propanediol production process by Clostridium butyricum through repeated batch fermentation coupled with activated carbon adsorption.

1,3-propanediol production by Clostridium butyricum is a low productivity process due to the long time seed cultivation and thus hinders its industrial scale production. In the present study, repeated batch fermentation coupled with activated carbon adsorption strategy was first established which conduced not only to saving the time of seed cultivation and enhancing the productivity, but also to reducing the costs for the seed cultivation to achieve the purpose of 1,3-propanediol continuous production. The concentration of 1,3-propanediol from first to fourth cycle was 42.89, 45.78, 44.48, 42.39 (g/L), and the corresponding volumetric productivity was 2.14, 1.91, 1.85, 2.12 (g/L · h-1 ) respectively. More importantly, a relatively complete schematic diagram of the proposed metabolic pathways was firstly mapped out based on the intracellular metabolites analysis through GC-MS. At the same time, metabolic pathway and principal components analyses were carried out to give us deep insight into metabolic state. Many metabolites occurred to response to the stress in Cycle II. Even resting body formed and lipid accumulated owing to the worsening environment in the group without activated carbon in Cycle III. Thus, it demonstrated that activated carbon provided a favorable microenvironment for Clostridium butyricum in the repeated batch fermentation process to achieve the purpose of 1,3-propanediol continuous production.

An efficient multi-stage fermentation strategy for the production of microbial oil rich in arachidonic acid in Mortierella alpina.

BACKGROUND: Fungal morphology and aeration play a significant role in the growth process of Mortierella alpina. The production of microbial oil rich in arachidonic acid (ARA) in M. alpina was enhanced by using a multi-stage fermentation strategy which combined fed-batch culture with precise control of aeration and agitation rates at proper times. RESULTS: The fermentation period was divided into four stages according to the cultivation characteristics of M. alpina. The dissolved oxygen concentration was well suited for ARA biosynthesis. Moreover, the ultimate dry cell weight (DCW), lipid, and ARA yields obtained using this strategy reached 41.4, 22.2, 13.5 g/L, respectively. The respective values represent 14.8, 25.8, and 7.8% improvements over traditional fed-batch fermentation processes. CONCLUSIONS: This strategy provides promising control insights for the mass production of ARA-rich oil on an industrial scale. Pellet-like fungal morphology was transformed into rice-shaped particles which were beneficial for oxygen transfer and thus highly suitable for biomass accumulation.

Lipid Fraction and Intracellular Metabolite Analysis Reveal the Mechanism of Arachidonic Acid-Rich Oil Accumulation in the Aging Process of Mortierella alpina.

The mechanism of arachidonic acid (ARA) content increase during aging of Mortierella alpina was elucidated. Lipid fraction analysis showed that ARA content increased from 46.9% to 66.4% in the triacylglycerol (TAG) molecule, and ARA residue occupation increased in the majority of TAG molecules during the aging process. For the first time, intracellular metabolite analysis was conducted to reveal the pathways closely associated with ARA biosynthesis during aging. The main reason for the increased ARA content was not only at the expense of other fatty acids degradation but also at the expense of further ARA biosynthesis during aging. Furthermore, translocation played a vital role in ARA redistribution among the glycerol moiety, and mycelium did not die immediately with key pathways activated to maintain a relatively stable intracellular environment. This study lays a foundation for further improvement of ARA content in the oil product obtained from M. alpina.

Efficient arachidonic acid-rich oil production by Mortierella alpina through a repeated fed-batch fermentation strategy.

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a long fermentation period needed process due to the low growth rate of the filamentous fungus used. This causes the low productivity of ARA-rich oil and hinders its industrial mass scale production. In the present study, different fed-batch strategies were conducted to shorten the fermentation period. The result showed that compared with the batch culture, the fermentation period was shortened from 7days to 5days with the productivity of ARA-rich oil increased from 0.9g/(L·d) to 1.3g/(L·d) by using the fed-batch fermentation strategy. Furthermore, repeated fed-batch fermentation strategy was adopted to achieve the purpose of continuous production. By using this strategy, the fermentation period was shortened from 40days to 26days in a four cycle repeated fed-batch fermentation. This strategy proved to be convenient and economical for ARA-rich oil commercial production process.

Efficient arachidonic acid-rich oil production by Mortierella alpina through a three-stage fermentation strategy.

A three-stage fermentation strategy was designed for efficient arachidonic acid (ARA)-rich oil production by Mortierella alpina. The process at different stages by changing the components of medium was investigated. In the first stage, mycelia were inoculated in a nutrient-rich medium for rapid propagation. In the second stage, mycelia were collected and then cultivated in glucose solution to achieve high cellular lipid contents. In the third stage, mycelia were cultured in a glucose-absent medium to obtain rapid ARA accumulation. Using this fermentation strategy, high dry cell weight, lipid, and ARA concentration reached 41.6, 26.6, and 11.4 g/L, respectively. The results demonstrated that mycelia propagation, lipid biosynthesis, and ARA accumulation process can be significantly spatially separated, allowing further optimization to improve the efficiency of each stage. This was the first report of using a three-stage fermentation strategy for ARA-rich oil production, and it could be applied to other similar oleaginous microorganisms to obtain high related polyunsaturated fatty acids accumulation.

Arachidonic acid-rich oil production by Mortierella alpina with different gas distributors.

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a high oxygen demand and shear-sensitive process. In the aerobic fermentation process, oxygen supply is usually a limiting factor owing to the low solubility of oxygen in the fermentation broth. Two kinds of perforated ring gas distributors and a novel microporous ceramic membrane gas distributor were designed and applied to improve oxygen supply. With the decrease of the orifice diameter of perforated ring gas distributors, dry cell weight (DCW), lipids concentration, and ARA content in total fatty acid increased from 17.86 g/L, 7.08 g/L, and 28.08 % to 25.67 g/L, 11.94 g/L, and 36.99 %, respectively. Furthermore, the effect of different dissolved oxygen (DO) on ARA-rich oil production with membrane gas distributor was also studied. The maximum DCW, lipid concentration, and ARA content using membrane gas distributor with DO controlled at 40 % reached 29.67 g/L, 16.74 g/L, and 49.53 %, respectively. The ARA titer increased from 1.99 to 8.29 g/L using the membrane gas distributor to substitute the perforated ring gas distributor. In the further experiment, a novel tubular titanium metal membrane gas distributor was successfully applied in a 7,000 L bioreactor and the results demonstrated that membrane gas distributor was industrially practical.

Improving the specific synthetic activity of a penicillin g acylase using DNA family shuffling.

Penicillin G Acylas (PGA) of Providencia rettgeri (ATCC 25599) was evolved using a modified DNA family shuffling method. The identity of pga genes from Escherichia coli, Kluyvera citrophila and Providencia rettgeri ranges from 62.5% to 96.9%. The pga genes from above three species were recombined and shuffled to create interspecies pga gene fusion libraries. By substituting assembled chimaeras for corresponding region of pETPPGA, different recombinants were constructed and expressed in E. coli JM109(DE3). Mutants with obvious beta-lactam synthetic activity were selected from the plates and the ratios of synthesis to hydrolysis (S/H) were determined subsequently. It was shown that the primary structures of selected positives exhibited significant diversity among each library. The best mutant possessed 40% higher synthetic activity than the wild type enzyme of PrPGA. It was further proved in this study that the domain of alpha subunit contributed much more to improve the specific activity of synthesis. Results showed a recombinant PGA with higher synthetic activity was acquired by the method of DNA shuffling.