Nitrogen Source Concentration Research Articles

A novel fungal and bacterial consortium promotes the degradation of rice straw: Conditions optimization and degradation properties

The rapid development of agriculture has led to the production of a large amount of crop straw, necessitating effective strategies for its management. Microbial degradation offers a promising method. In this study, a novel microbial consortium composed of Phanerochaete chrysosporium, Aspergillus niger, and Streptomyces griseorubens, known for their robust lignocellulose degradation capabilities, was constructed for rice straw degradation. The establishment of this microbial consortium was based on the growth curve and antagonistic tests. Orthogonal optimization revealed that Streptomyces griseorubens played a predominant role in the degradation of rice straw. The optimal degradation conditions were determined as follows: nitrogen source concentration of 2.5 gL−1, material-liquid ratio of 40 g L−1, inoculum size of 3%, and pH value of 9. Under these conditions, the degradation efficiency reached 42% within 15 days. The decomposition of lignocellulosic components in the straw was confirmed through various characterization methods. Additionally, as the degradation process progressed, there was a noticeable decrease in protein-like substances and an increase in humic acid-like substances in the degradation solution.

Production of arabitol from glycerol by immobilized cells of Wickerhamomyces anomalus WC 1501.

Polyalcohols such as arabitol are among the main targets of biorefineries aiming to upcycle wastes and cheap substrates. In previous works Wickerhamomyces anomalus WC 1501 emerged as an excellent arabitol producer utilizing glycerol. Arabitol production by this strain is not growth associated, therefore, in this study, pre-grown cells were entrapped in calcium alginate beads (AB) and utilized for glycerol transformation to arabitol. Flasks experiments aimed to assess the medium composition (i.e., the concentration of inorganic and organic nitrogen sources and phosphates) and to establish the appropriate carrier-to-medium proportion. In flasks, under the best conditions of ammonium limitation and the carrier:medium ratio of 1:3 (w/v), 82.7g/L glycerol were consumed in 168h, yielding 31.2g/L arabitol, with a conversion of 38% and volumetric productivity of 186mg/mL/h. The process with immobilized cells was transferred to laboratory scale bioreactors with different configurations: stirred tank (STR), packed bed (PBR), fluidized bed (FBR), and airlift (ALR) bioreactors. The STR experienced oxygen limitation due to the need to maintain low stirring to preserve AB integrity and performed worse than flasks. Limitations in diffusion and mass transfer of oxygen and/or nutrients characterized also the PBR and the FBR and were partially relieved only in ALR, where 89.4g/L glycerol were consumed in 168h, yielding 38.1g/L arabitol, with a conversion of 42% and volumetric productivity of 227mg/mL/h. When the ALR was supplied with successive pulses of concentrated glycerol to replenish the glycerol as it was being consumed, 117g/L arabitol were generated in 500h, consuming a total of 285g/L glycerol, with a 41% and 234mg/L/h. The study strongly supports the potential of W. anomalus WC 1501 for efficient glycerol-to-arabitol conversion using immobilized cells. While the yeast shows promise by remaining viable and active for extended periods, further optimization is required, especially regarding mixing and oxygenation. Improving the stability of the immobilization process is also crucial for reusing pre-grown cells in multiple cycles, reducing dead times, biomass production costs, and enhancing the economic feasibility of the process.

Efficient production of butyl butyrate from mannitol by engineered Clostridium tyrobutyricum

Butyl butyrate has a wide range of applications as flavors, solvents, and biofuels. Green production of butyl butyrate by microorganism fermentation in an industry level attracts great attention on the basis of bio-refinery. In this study, a dark horse Clostridium tyrobutyricum was employed for de novo production of butyl butyrate. Herein, reinforcement of EMP and butyl butyrate fermentation pathways (e.g., pfkA, pykA, crt-hbd-bcd/etfAB, adhE2 and VAAT) and regulation of carbon distribution towards butyl butyrate (e.g., CAT1 inactivation or overexpression of NADPH-dependent hbd1) were conducted in C. tyrobutyricum (MUV3) to enhance butyl butyrate production. The outstanding strain MV12 generated 1.8-fold butyl butyrate production compared to the initial strain MUV3. Subsequently, organic nitrogen source concentration was optimized, 21.1 g/L and 30.1 g/L butyl butyrate were obtained in 37 °C and 25 °C, respectively. Ultimately, fed-batch with two-stage temperature shift fermentation was performed for high titer and yield of butyl butyrate production (142.6 g/L titer and 0.28 mol/mol yield) from mannitol. Hence this study presented a microbial fermentation platform for butyl butyrate or other value-added chemicals production from renewable resources.

Effects of eutrophication on the horizontal transfer of antibiotic resistance genes in microalgal-bacterial symbiotic systems

Overloading of nutrients such as nitrogen causes eutrophication of freshwater bodies. The spread of antibiotic resistance genes (ARGs) poses a threat to ecosystems. However, studies on the enrichment and spread of ARGs from increased nitrogen loading in algal-bacterial symbiotic systems are limited. In this study, the transfer of extracellular kanamycin resistance (KR) genes from large (RP4) small (pEASY-T1) plasmids into the intracellular and extracellular DNA (iDNA, eDNA) of the inter-algal environment of Chlorella pyrenoidosa was investigated, along with the community structure of free-living (FL) and particle-attached (PA) bacteria under different nitrogen source concentrations (0–2.5 g/L KNO3). The results showed that KR gene abundance in the eDNA adsorbed on solid particles (D-eDNA) increased initially and then decreased with increasing nitrogen concentration, while the opposite was true for the rest of the free eDNA (E-eDNA). Medium nitrogen concentrations promoted the transfer of extracellular KR genes into the iDNA attached to algal microorganisms (A-iDNA), eDNA attached to algae (B-eDNA), and the iDNA of free microorganisms (C-iDNA); high nitrogen contributed to the transfer of KR genes into C-iDNA. The highest percentage of KR genes was found in B-eDNA with RP4 plasmid treatment (66.2%) and in C-iDNA with pEASY-T1 plasmid treatment (86.88%). In addition, dissolved oxygen (DO) significantly affected the bacterial PA and FL community compositions. Nephelometric turbidity units (NTU) reflected the abundance of ARGs in algae. Proteobacteria, Cyanobacteria, Bacteroidota, and Actinobacteriota were the main potential hosts of ARGs. These findings provide new insights into the distribution and dispersal of ARGs in the phytoplankton inter-algal environment.

Bioreactor study of the metabolic repertoire and morphology of actinomycete Streptomyces rimosus ATCC 10970 under various initial concentrations of carbon and nitrogen sources

This work aims at investigating the influence of the initial concentrations of carbon (glucose) and organic nitrogen (yeast extract) sources on Streptomyces rimosus ATCC10970 secondary metabolism in the stirred tank bioreactors. Additionally, glucose utilisation, biomass formation, pH, redox potential and dissolved oxygen levels, and the morphological development of S. rimosus pseudomycelium were studied. Eighteen secondary metabolites were detected by mass spectrometry and identified with the use of the authentic standard, or putatively with the use of literature and database of secondary metabolites. Varied initial yeast extract concentration acted much stronger on the formation of secondary metabolites than glucose did. For example, oxytetracycline was not biosynthesised at high yeast extract concentration while the formation of three other metabolites was enhanced under these conditions. In the case of glucose its increasing initial concentration led to higher secondary metabolite levels with the exception of an unnamed angucycline. High initial yeast extract concentration also drastically changed S. rimosus pseudomycelial morphology from the pelleted to the dispersed one. Ultimately, the cultivation media with the varied initial levels of carbon and nitrogen sources were proved to have the marked effect on S. rimosus secondary metabolism and to be the simplest way to either induce or block the formation of the selected secondary metabolites.

Optimized carotenoid production and antioxidant capacity of Gordonia hongkongensis.

The current emphasis within the cosmetic market on sustainable ingredients has heightened the exploration of new sources for natural, active components. Actinomycetota, recognized for producing pigments with bioactive potential, offer promising functional cosmetic ingredients. This study aimed to optimize pigment and antioxidant metabolite production from the Gordonia hongkongensis strain EUFUS-Z928 by implementing the Plackett-Burman experimental design and response surface methodology. Extracts derived from this strain exhibited no cytotoxic activity against human primary dermal fibroblast (HDFa, ATCC® PCS-201-012™, Primary Dermal Fibroblast; Normal, Human, Adult). Eight variables, including inoculum concentration, carbon and nitrogen source concentration, NaCl concentration, pH, incubation time, temperature, and stirring speed, were analyzed using the Plackett-Burman experimental design. Subsequently, factors significantly influencing pigment and antioxidant metabolite production, such as temperature, inoculum concentration, and agitation speed, were further optimized using response surface methodology and Box-Behnken design. The results demonstrated a substantial increase in absorbance (from 0.091 to 0.32), DPPH radical scavenging capacity (from 27.60% to 84.61%), and ABTS radical scavenging capacity (from 17.39% to 79.77%) compared to responses obtained in the isolation medium. The validation of the mathematical model accuracy exceeded 90% for all cases. Furthermore, liquid chromatography coupled with mass spectrometry (LC-MS) facilitated the identification of compounds potentially responsible for enhanced pigment production and antioxidant capacity in extracts derived from G. hongkongensis. Specifically, six carotenoids, red-orange pigments with inherent antioxidant capacity, were identified as the main enhanced compounds. This comprehensive approach effectively optimized the culture conditions and medium of a G. hongkongensis strain, resulting in enhanced carotenoid production and antioxidant capacity. Beyond identifying bioactive compounds and their potential cosmetic applications, this study offers insights into the broader industrial applicability of these extracts. It underscores the potential of G. hongkongensis and hints at the future utilization of other untapped sources of rare actinomycetes within the industry.

The Liquid Fermentation Process for Mycelia of Poria cocos (Agaricomycetes) by Single-Factor Experimentation and Response Surface Methodology.

Liquid fermentation could yield substantial mycelia mass and valuable secondary metabolites in large-scale production within a short, fermented duration. The liquid fermented process of mycelia of Poria cocos was optimized using a combination of single-factor experimentation and response surface methodology (RSM) to obtain more extract of P. cocos. The optimal conditions were determined as follows: The carbon source concentration at 1%, the nitrogen source concentration at 1%, the inoculum volume at 7% and a culture time of 9 d. Under these conditions, the ethyl acetate extract mass of P. cocos mycelia reached 0.0577 ± 0.0041 mg. There were significant interactions between nitrogen source concentration and cultivation time. The predicted values by the mathematical model based on the response surface analysis showed a close agreement with experimental data.

Sustainable approach for the degradation of Contrast dye Evans blue by Enterobacter cloacae strain SD4-1

BackgroundDischarge of dye complexes through industrial effluents continues to be a never-ending problem for the environment. Several degradation techniques have been experimented with, of which utilizing biological entities for the effective degradation of these dye molecules is extensively studied and still being explored. The present study focuses on (i) isolation of bacterial strains from the contaminated agricultural lands (ii) screening the isolates for their decolourization efficiency of Evans blue dye (iii) optimization of culture parameters such as temperature, pH, concentration of dye and nitrogen source for effective decolourization (iv) analysing the effect of degradation of diazo dye Evan's blue by the selected strain using GCMS analysis. MethodsThe bacterial strains were isolated from contaminated agricultural lands in Chennai, Tamil Nadu, India. The collected samples were aseptically transferred to the laboratory and stored for further studies. The collected samples were serially diluted, and the colonies with distinct morphology were pure-cultured. The isolated strains were studied for their efficiency to decolourize the dye Evans blue dye. The strain with higher decolourization efficiency was selected and optimized for parameters such as temperature, pH, nitrogen source and concentration of dye. The degradation of dye by the isolate was analysed by GCMS. Significant FindingsThe investigation unveiled that the strain SD4-1 of Enterobacter cloacae effectively decolorizes Evans blue dye at 37°C and pH 6.0, utilizing Sodium nitrate as the nitrogen source. SD4-1 exhibited an impressive dye decolourization efficiency of approximately 75%. The comprehensive GCMS analysis verified the bacterium's capability to fully degrade the azo dye Evans blue. Consequently, this study concludes that the SD4-1 Enterobacter cloacae strain holds promising potential for the efficient biodegradation of Evan's blue dye. Its application as a highly effective biological agent in the practical removal of dyes from textile industries is envisaged. Notably, this organism demonstrates superior performance in comparison to other studies where a mixture of dyes or consortia of microorganisms were employed for dye decolourization.

CYTOTOXIC EFFECT OF ASPARAGINASE PRODUCE FROM E.COLI AND CONJUGATED WITH ZNO NANOPARTICLES ON CANCER CELL LINES

This study was aimed to examine the anticancer activity of fabricated ZnONPs- L-ASNase conjugate against cancer and normal cell lines. Total of 127 bacterial isolates were taken from clinicalandenvironmentalsamples then identified as E.coli.In this work, 88 isolates were identified as E.coli.L-asparaginase produced from E.coli were screened¸clinical Isolates of E.coli from urine sample have the potential to produce asparaginase that is responsible for cytotoxicity¸the optimal culture conditions for the development of the enzyme L-asparaginase were determined to be 37ºC and pH8. Nitrogen sourceconcentrations at 0.9gm/ml were discovered to maximize enzyme production. L-asparaginase then purified by two steps that includeion exchange column and gel filtration. Purification and recovery percentage for L-ASNase was 6.21%, 10.34% respectively. Effective conjugation was approved by UV-visible spectroscopy. ZnO nanoparticles exhibit absorbance peaks approximately 200-300 nm. XRD analysis confirmed the crystallite size of the ZnO nanoparticles was observed to be 18.4 nm. The activity of L-ASNase in the ZnONPs-L-ASNasewas detected by a direct nesslerization method and the findings showed a substantial increase L-ASNase specific activity within the conjugate of ZnONPs-L-ASNase in comparison to its free state. The ZnONPs-L-ASNase conjugate showed the greatest and most substantial cytotoxic action against A375 with survival rate at 400 µg /mL about 45.8± 2.3. The IC50 value of 44.69 µg/ mL of ZnONPs- L-ASNase was calculated for A375 cells. In comparison, all substances exhibited weak to moderate cytotoxicity when tested against the normal cell line WRL-68. The ZnONPs-L-ASNase combination therapy resulted in a higher cytotoxic effect. This action could be attributed to the synergetic effects of both L-ASNase and the conjugated molecules ZnONPs.

Bioconversion of glycerol into polyhydroxyalkanoates through an atypical metabolism shift using Priestia megaterium during fermentation processes: A statistical analysis of carbon and nitrogen source concentrations

Polyhydroxyalkanoates (PHA) are bioplastics which are well known as intracellular energy storage compounds and are produced in a large number of prokaryotic species. These bio-based inclusions are biodegradable, biocompatible and environmental friendly. Industrial production of, short chain and medium chain length PHA, involves the use of microorganisms and their enzymes. Priestia megaterium previously known as Bacillus megaterium is a well-recognized bacterium for producing short chain length PHA. This study focuses to characterize this bacterium for the production of medium chain length PHA, and a novel blend of both types of monomers having enhanced properties and versatile applications. Statistical analyses and simulations were used to demonstrate that cell dry weight can be derived as a function of OD600 and PHA content. Optimization of growth conditions resulted in the maximum PHA production as: 0. 05 g. g-x. H−1, where the rate of PHA production was 0.28 g L−1. H−1 and PHA concentration was 4.94 g. L−1. This study also demonstrated FTIR to be a semi quantitative tool for PHA production. Moreover, conversion of scl-PHA to mcl-PHA with reference to time intermissions using GC-FID are shown.

Soft sensor based on Raman spectroscopy for the in-line monitoring of metabolites and polymer quality in the biomanufacturing of polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHA) are among the most promising bio-based alternatives to conventional petroleum-based plastics. These biodegradable polyesters can in fact be produced by fermentation from bacteria like Cupriavidus necator, thus reducing the environmental footprint of the manufacturing process. However, ensuring consistent product quality attributes is a major challenge of biomanufacturing. To address this issue, the implementation of real-time monitoring tools is essential to increase process understanding, enable a prompt response to possible process deviations and realize on-line process optimization. In this work, a soft sensor based on in situ Raman spectroscopy was developed and applied to the in-line monitoring of PHA biomanufacturing. This strategy allows the collection of quantitative information directly from the culture broth, without the need for sampling, and at high frequency. In fact, through an optimized multivariate data analysis pipeline, this soft sensor allows monitoring cell dry weight, as well as carbon and nitrogen source concentrations with root mean squared errors (RMSE) equal to 3.71, 7 and 0.03 g/L, respectively. In addition, this tool allows the in-line monitoring of intracellular PHA accumulation, with an RMSE of 14 gPHA/gCells. For the first time, also the number and weight average molecular weights of the polymer produced could be monitored, with RMSE of 8.7E4 and 11.6E4 g/mol, respectively. Overall, this work demonstrates the potential of Raman spectroscopy in the in-line monitoring of biotechnology processes, leading to the simultaneous measurement of several process variables in real time without the need of sampling and labor-intensive sample preparations.

Influencia de la variación de nutrientes en la alimentación sobre la acumulación de AbrB, esporulación y expresión de cry1Ac durante cultivos por lote alimentado de Bacillus thuringiensis

The biological control agent Bacillus thuringiensis (Bt) coordinates metabolic and gene regulation functions to enter to the differentiation process for the synthesis of spores and Cry toxin proteins. The master regulator for sporulation initiation is Spo0A, while AbrB regulator coordinate cellular functions during vegetative and transition growth. To understand how differentiation is modified during fed-batch cultures (FBC) of Bt under variable nutrient conditions, we conducted fermentations with two different nutrient concentration and feeding times using an industrial medium. Since sporulation efficiency has been lower in FBCs than in batch cultures, AbrB was monitored by Western blot during these cultures. Kinetic parameters were used to stablish timing of cell differentiation process, and fermentations were monitored on-line using impedance spectroscopy. AbrB was accumulated during the entire fed-batch culture, when a medium with the highest concentration of carbon and nitrogen sources (FBC1) were fed. Under these conditions, sporulation efficiency and cry1Ac expression were negatively affected, compared to the wild-type strain. Conversely, when the feeding concentration was only half of FBC1, AbrB accumulates in minor proportion, and sporulation efficiency and cry1Ac expression increased (FBC2). These results suggest that variable nutrient conditions can cause an imbalance in the accumulation of transition state regulators such as AbrB during FBCs, which can be detrimental to produce spores and Cry proteins, that are the main Bt products for biological control of agricultural pests.

Optimising fertigation of hydroponically grown sowthistle (Sonchus oleraceus L.): The impact of the nitrogen source and supply concentration

The efficient use of water and nutrients in leafy vegetable crops can be improved by effective water management, whereas the form of the supplied nitrogen may affect their quality and productivity. Sowthistle (Sonchus oleraceus L.), a widespread weed collected and consumed as a wild vegetable in the past, currently attracts a high interest for commercial cultivation due to its high nutritional value. The present study evaluated the effects of different nitrogen concentrations: N50: 50 mg L−1; (N 3.6 mmol L−1), N100: 100 mg L−1; (N 7.1 mmol L−1), N200: 200 mg L−1; (N 14.3 mmol L−1), and N300: 300 mg L−1; (N 21.4 mmol L−1) and different ammonium/total-N ratios (Nr: 0.01, 0.05, 0.10, 0.15) on growth, physiological parameters, antioxidant capacity and nutrient accumulation in the different plant parts of sowthistle, grown in Nutrient Film Technique hydroponic system. Total N was ammonium N plus nitrate N. Plant growth, total phenols, and antioxidant capacity were higher at the two intermediate N concentrations, whereas flavonoid and nitrogen content, as well as irrigation water productivity increased only with 200 mg N L−1 (N 14.3 mmol L−1), compared to the other three N concentrations. The accumulation of nitrogen in leaves and roots was lower, while leaf stomatal conductance was enhanced by increased N concentrations in the nutrient solution. Higher Nr ratios than ≥ 0.05 decreased plant dry matter, total phenols, flavonoids, and antioxidant capacity in leaf extracts, and negatively affected nitrogen translocation from roots to leaves. Sowthistle plants treated with a Nr of 0.05 exhibited a less intense oxidative stress, with decreased lipid peroxidation and hydrogen peroxide production, and increased superoxide dismutase and catalase activities, compared to those treated with higher Nr ratios. Increased Nr resulted in the accumulation of phosphorus and magnesium in leaves while the highest irrigation water productivity was obtained in plants grown with a Nr of 0.05. In conclusion, to increase yield, nutritional value and efficiency of water and nitrogen use in sowthistle grown in closed hydroponic systems, a N concentration of 200 mg L−1 of N (N 14.3 mmol L−1) and a Nr of 0.05 are suggested.

Optimization of polyhydroxyalkanoate production in Halomonas sp. YLGW01 using mixed volatile fatty acids: a study on mixture analysis and fed-batch strategy

Polyhydroxyalkanoate (PHA) is one of the most promising materials for replacing petroleum-based plastics, and it can be produced from various renewable biomass sources. In this study, PHA production was conducted using Halomonas sp. YLGW01 utilizing mixed volatile fatty acids (VFAs) as carbon sources. The ratio and concentration of carbon and nitrogen sources were optimized through mixture analysis and organic nitrogen source screening, respectively. It was found that the highest cell dry weight (CDW) of 3.15 g/L and PHA production of 1.63 g/L were achieved when the ratio of acetate to lactate in the mixed VFAs was 0.45:0.55. Furthermore, supplementation of organic nitrogen sources such as soytone resulted in a ninefold increase in CDW (reaching 2.32 g/L) and a 22-fold increase in PHA production (reaching 1.60 g/L) compared to using inorganic nitrogen sources. Subsequently, DO-stat, VFAs consumption rate stat, and pH–stat fed-batch methods were applied to investigate and evaluate PHA productivity. The results showed that when pH–stat-based VFAs feeding was employed, a CDW of 7 g/L and PHA production of 5.1 g/L were achieved within 68 h, with a PHA content of 73%. Overall, the pH–stat fed-batch strategy proved to be effective in enhancing PHA production by Halomonas sp. YLGW01 utilizing VFAs.

Efficient production of biomass and exopolysaccharide from P. ostreatus and physio-chemical characterization of biomass powder

Mycelium biomass and exopolysaccharides (EPS) from Pleurotus ostreatus origin have gained peculiar attention because of their myriad applications in the food and pharmaceutical industries. Mycelial growth is greatly dependent on fermentation media and physical conditions. This study aims to optimize mycelium biomass and EPS using one factor at a time (OFAT) approach. The current study investigates the effect of the concentration of various carbon, nitrogen sources, metal ions, and cell numbers in inoculum for biomass and EPS production. The results revealed that the 5% w/v malt extract and 2% w/v glucose combined with 0.05% w/v of Mg+ ions gave the maximum yield of biomass and EPS as 31.55 g/L and 6.65 g/L respectively. A high protein and total phenolics content have been obtained in the supernatant of media as 5.178 g/L and 104.18 mg GAE/g dry weight respectively. Furthermore, the obtained lyophilized biomass powder was investigated by FTIR which showed the presence of proteins and polysaccharides. The morphology and elemental composition were observed by SEM-EDS. XRD pattern showed the amorphous nature of biomass but the presence of chitin was also observed.

Pengaruh Perbedaan Jenis dan Konsentrasi Sumber Nitrogen (NaNO3 dan urea) terhadap Produksi Biomasa Spirulina Platensis

The goal of this study was to investigate the effect of the concentration and the type of nitrogen sources in the cultivation medium on the production of biomass and biochemical content (carbohydrates, proteins, fats) in the Spirulina platensis biomass. Types of nitrogen sources included NaNO3 and urea, where the concentrations were varied from 0.5 to 3.5 g/L. The results showed that the use of urea tended to produce more S. platensis biomass than the use of NaNO3. The best concentrations that produced the highest biomass were NaNO3 2.5 g/L (biomass 0.6745 g/L) and urea 0.5 g/L (biomass 0.7158 g/L). The use of urea also tended to produce a higher specific growth rate and a shorter doubling time than the use of NaNO3. The high concentration of nitrogen sources caused the pH of the medium to increase more rapidly due to the accumulation of ammonium in the medium. Using NaNO3 can produce biomass with a higher protein content (28.34-36.98%) than using urea (25.86-33.52%).

Utilization of non-pathogenic bacteria to obtain optimum biofilm production for beneficial applications.

Depending on the bacteria embedded in the extracellular polymeric layer, biofilms can be advantageous or harmful. The isolated strains used in this investigation are already established to be beneficial biofilm-producing bacteria. In order to use them effectively in various domains, it is necessary to characterize them and understand their ideal physiological characteristics for maximumbiofilm growth. This study used genome sequence analysis to identify and characterize strains isolated from water samples in Raipur, Chhattisgarh, India. The nucleotide sequences were submitted to NCBI GenBank under the accession numbers Bacillus tequilensis (MN889418) and Pseudomonas beteli (MN889419) and the strains were further characterized using some advanced techniques (phase contrast microscopy, Raman spectroscopy, Fourier-transform infrared spectroscopy, and scanning electron microscope). For maximum biofilm formation by isolated bacterial strains, many physiochemical factors including incubation duration, temperature, pH, carbon source concentration, and nitrogen source concentration were further examined and optimized. The fact that these non-pathogenic strains were found in public water supplies is another important part of this research because there is a chance that they could change into pathogenic state in future and cause disease in humans.

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization

BackgroundRaw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value.ResultsIn this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production.ConclusionsThis study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.

Effects of dissolved organic carbon and total ammonia nitrogen concentrations with the same DOC/TAN on biofloc performance

The regulation of the carbon-to‑nitrogen ratio (C/N) is at the core of biofloc technology. This study compared the performance of bioflocs under different dissolved organic carbon (DOC) and total ammonia nitrogen (TAN) concentrations but the same DOC/TAN ratio. Dissolved organic carbon and total ammonium nitrogen were set at 200 mg/L and 10 mg/L (Group 200/10), 100 mg/L and 5 mg/L (Group 100/5), and 50 mg/L and 2.5 mg/L (Group 50/2.5), respectively. The TAN removal rates of groups A, B, and C were 1.89 ± 0.02 mg N/g TSS·h, 1.63 ± 0.03 mg N/g TSS·h, and 1.32 ± 0.02 mg N/g TSS·h, respectively, and the differences were significant (P < 0.05). At the phylum level, the microbial community structure was dominated by Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, and Planctomycetes. At the genus level, Gemmobacter, Hydrogenophaga, and Microbacterium were the dominant genera. Most of bacteria community differed significantly among the three groups. As the concentration of carbon and nitrogen sources increased, the Shannon index showed a decrease in bacterial diversity, and the number of heterotrophic bacteria significantly increased. The crude protein content of flocs in group A ((29.22 ± 0.20)%) was significantly lower than that in groups B ((30.56 ± 0.32)%) and C ((30.83 ± 0.20)%). In groups A and B with higher carbon and nitrogen concentrations, the copy numbers of genes related to nitrification were relatively low, but the copy numbers of genes related to denitrification were relatively high. Therefore, it can be concluded that with a ratio of 20 of DOC/TAN, increasing DOC and TAN concentrations led to a significant increase in TAN removal capacity, heterotrophic bacteria count, denitrification function gene copies, and crude protein content of bioflocs, and a decrease in bacterial diversity, nitrification function gene copies, and C/N of bioflocs (P < 0.05). These results suggest that the influence of DOC and TAN concentrations on bioflocs performance may be more significant than that of DOC/TAN, and should be fully considered in practical bioflocs technology systems.

Experimental Study on the Effective Production of Biocement for Soil Solidification and Wind Erosion Control

Biocement can be achieved through the microbially induced carbonate precipitation (MICP) process. Such a method can potentially be utilized as an eco-friendly method for civil and environmental engineering applications such as soil ground improvement and wind erosion control of surface soil. In this method, one key step is the effective production of ureolytic bacteria. In previous laboratory and field studies, the cultivation and production of the bacteria used for the MICP were usually expensive and time-consuming. The purpose of this study was to optimize the cultivation method of the ureolytic bacteria (Sporosarcina pasteurii), and soil stabilization tests were conducted to verify the effectiveness of the cultured bacteria used to strengthen soil against the wind-induced erosion. Bacterial cultivation methods were studied by investigating the effects of different cultivation media and conditions. Testing variables included the types and concentrations of nitrogen sources (urea or NH4Cl), pH values (7.5–9.5), cultivation conditions (batch or chemostat condition), and different carbon sources. It was found that, with the same amount of nitrogen source, the test with pure urea had the highest biomass yield, urease activity, and specific urease activity than the other tests with pure NH4Cl or both NH4Cl and urea. The use of urea as the nitrogen source in the media also led to an increase in pH, which was not found in the test with pure NH4Cl. As for the factor of urea concentration, the tests with a higher urea concentration had a higher biomass yield, urease activity, and pH. The factor of pH values also played an important role. The test with an 8.5 initial pH value had a higher biomass yield, urease activity, and specific urease activity than the tests with 7.5 and 9.5 initial pH values. In the chemostat condition, the ureolytic bacteria could be effectively produced with urease activity up to 7 mmol/L/min, as compared with around 12 mmol/L/min activity in the batch condition. Thus, the optimum nitrogen source, pH value, and cultivation condition for the cultivation of Sporosarcina pasteurii was urea, 8.5, and batch condition, respectively. In addition, when soybean milk powder or milk powder was used as the carbon source, the urease activity was around 2.5 mmol/L/min, which is also high enough to be used for biocement.