Culturable Bacterial Population Research Articles

Bioremediation of a crude oil contaminated soil using water lettuce (<i>Pistia stratiotes</i>)

Laboratory-scale studies were carried out using a nutrient formula produced from Pistia stratiotes plant to achieve bioremediation of crude oil impacted loamy soil. This study assessed the efficacy of powdered Pistia stratiotes as potential bio stimulant in the remediation of crude oil contaminated soil using three test treatments (20ml, 60ml and 100ml) at three test concentrations (20g, 40g and 60g) of nutrient and a control (0g). The remediation process was monitored by assaying the total hydrocarbon content (THC) and soil pH before and after amendment with the powdered P. stratiotes for 90 days. The result showed increase in soil pH and THC in comparison with the control due to soil contamination by crude oil. However, there was a significant reduction (p < 0.05) in soil pH and THC with the introduction of powdered P. stratiotes at different concentrations. Contaminated soil amended with 40g of P. stratiotes had the highest THC loss of (30%) on the final day of remediation. In a 90 day study, the control set-up had its total culturable heterotrophic bacterial population of the P. stratiotes recipe increase from 1.4×107 Cfu/g to 2.8×107 Cfu/g, while the total culturable heterotrophic fungal count in the P. stratiotes treatment ranged from 7.0×104 Cfu/g to 4.0×104 Cfu/g. Statistical analyses showed significant difference at p ≤ 0.05 level for the two conditions (Pistia stratiotes nutrient treated soil and control). The total culturable hydrocarbon utilizing bacteria in P. stratiotes treated polluted soil increased from 1.0×106 Cfu/g to 6.8×105 Cfu/g, while the total culturable hydrocarbon utilising fungal counts increased significantly from 1.9×104 Cfu/g to 4.8×104 Cfu/g for P. stratiotes treatment. The results from this research demonstrated that powdered P. stratiotes is an effective agent that can be used to decrease the pH of contaminated soil and improve soil properties, hence aids in remediating a crude oil contaminated soil.

Análisis y caracterización de poblaciones bacterianas solubilizadoras de P en un ensayo de larga duración con diferentes secuencias de cultivo

It is well known that different soil use and management systems affect the abundance, activity and composition of the soil microbial community. In this work, the behavior of cultivable bacterial populations and, in particular, P (PSB) solubilizing bacteria in soil samples from a long-term trial with different culture sequences was studied. The results obtained show that the clearing and subsequent agricultural use after 11 years generated a decrease in the population of cultivable bacteria in general, and of P-solubilizing bacteria in particular, with respect to the pristine soil. Isolates with high P solubilization efficiency were obtained from the soils from the different rotations. These efficient isolates were taxonomically classified by 16S RNA analysis as belonging to the genera Bacillus, Paenibacillus, Pseudomonas and Xanthomonas. In particular, the characterization of the culture supernatants of the isolates Pseudomonas koreensis and Paenibacillus pabuli showed they are producers of organic acids. The combined inoculation tests of these two strains on maize plants in a culture chamber revealed a synergistic effect on the growth promotion of this species. The results presented here suggest that although PSB populations are more numerous in pristine soils, certain long-term crop rotations favor the increase of more efficient solubilizing bacteria, an aspect that should be considered when designing future search strategies for potential bioinoculants.

Sorption–Desorption of Imazamox and 2,4-DB in Acidic Mediterranean Agricultural Soils and Herbicide Impact on Culturable Bacterial Populations and Functional Diversity

In this study, we investigated the sorption–desorption behavior of imazamox (IMZ) and 2,4-DB (DB) in two typical acidic Mediterranean agricultural soils and the impact of these herbicides on culturable soil bacterial populations, enzyme activities and functional diversity when applied at concentrations higher than recommended doses (10×, 50×, 500×). Herbicide sorption was similar in both soils and IMZ was less retained compared to DB (~0.5 vs. 40 µg g−1 soil, respectively). IMZ desorption was remarkable (70–100%) while that of DB was more limited, i.e., ~40%. Three days after spiking (DAS), IMZ and DB significantly increased the number of soil-culturable heterotrophic bacteria, actinomycetes and Pseudomonas spp., soil respiration and the potential catabolic capacity of soil microbial communities. Soil dehydrogenase activity increased by ~56–70% in IMZ-treated soils while being reduced by ~33–41% in DB-treated ones. β-glucosidase activity showed a soil-dependent behavior, while the pattern of C source utilization suggested a change of soil microbial community structure after herbicide (especially DB) spiking. At 30 DAS, the herbicides’ impact on soil microorganisms, enzyme activity and functional diversity was still visible. Moreover, a toxic effect of DB (at 50× and 500×), but not IMZ, was recorded vs. Rhizobium sullae, the bacterial symbiont of Hedysarum coronarium. The obtained results indicated that IMZ and DB are poorly sorbed and highly desorbed by both soils. Moreover, at the tested concentrations, IMZ and DB can have short- and medium-term impacts on the microbial component and the related activity of the investigated soils, likely affecting a range of ecosystem services provided by soil microorganisms.

Effect of ZnO and CuO Nanoparticles on Culturable Bacterial Population, Microbial Biomass, and Enzyme Activities in Two Soil Types

Metal oxide nanoparticles are widely used in many agricultural, medical, and electronic products. This study was conducted to assess the effects of metal oxide nanoparticles on the total culturable soil bacterial population and enzymatic activities – namely, dehydrogenase (DHA), urease (UA), and microbial biomass (MCB) – using Philippine soils. The factors included the two soil types – Lipa clay loam (LCL) and Sariaya sandy loam (SSL) – and five treatments – namely, copper oxide bulk particles (bCuO), zinc oxide bulk particles (bZnO), copper oxide nanoparticles (CuONPs), zinc oxide nanoparticles (ZnONPs), and untreated control. The experiment was laid out in a split-plot completely randomized design over a 28- day incubation period under laboratory conditions. Results showed no significant effect on the culturable bacterial population. The DHA of ZnONPs-, bCuO-, and CuONPs-treated LCL was significantly reduced up to 7, 14, and 28 days after amendment (DAA), respectively. This result was probably due to the strongly acidic property, high level of organic matter (OM), low permeability due to particle size distribution, and low phosphate concentration of LCL. For UA, it was significantly reduced in LCL by CuONPs up to 28 DAA, perhaps due to the inactivation of soil UA. UA, however, was significantly increased by ZnONPs up to 28 DAA, presumably due to the release of intracellular urease from dead cells due to the toxic level of Zn2+. Soil MCB was significantly reduced at 7 DAA in CuONP-amended LCL and ZnONPs- amended SSL, whereas bCuO- and bZnO-amended LCL had a significant increase in MCB. The effect of CuONPs and ZnONPs on the parameters measured was influenced by soil type. In conclusion, CuONPs could pose a threat to soil microorganisms, especially in clayey soils.

Effects of Glyphosate and Butachlor Herbicides Concentrations on Resident Culturable Bacterial Populations from Surface Water Sources in Makurdi: A Microcosm Study

The effect of different concentrations of herbicides on bacterial isolates from various surface water sources was undertaken. The research was aimed at the isolation, identification and characterization of bacteria from selected surface water sources, in order to ascertain the effects of varying concentrations of herbicides on resident bacterial populations. Samples were obtained from five different surface water sources and sediments in Makurdi, using sterilized plastic containers. The herbicides used were Butachlor and Glyphosphate ammonium salt with Rid-over’s trade name (500, 250, 125, 62.5 and 31.5 mg). Both herbicides were spiked in the volume of water samples to reflect the recommendation of the manufacturer). The bacteriological analysis of the samples prior to spiking with the herbicides indicated that, Afuko stream had the highest total heterotrophic bacteria (THB) count of 4.55x107cfu/mL; River Benue 3.45x107cfu / mL; Akighir stream 3.0x107cfu / mL; whereas Mu river and Jamu stream had the lowest total heterotrophic bacteria count of 2.36x107cfu / mL and 2.31x107cfu/mL respectively. The isolates from the water sources in the order of highest percentage occurrences included; Escherichia coli 24(21.7%); Bacillus sp 22(21.2%); Salmonella sp 20(17.6%); Pseudomonas sp 17(15.0%); Proteus sp 16(14.1%) and Staphylococcus aureus 14 (12.3%) before spiking with the herbicides. The number of bacteria colonies increased as the days increased after spiking at different concentrations of the herbicides though colony counts were higher at lower concentrations than higher concentrations. The bacteria isolates have shown resistance to herbicides, which implies biogenic elements for their physiological processes.

Temporal Dynamics of Endogenous Bacterial Composition in Rice Seeds During Maturation and Storage, and Spatial Dynamics of the Bacteria During Seedling Growth.

Seed endophytes are of interest because they are believed to affect seed quality, and ultimately, plant growth and fitness. A comprehensive understanding of the assembly of the seed microbiome during seed development and maturation, the fate of microbes during storage, and the migration of microbes during seedling growth are still lacking. In this study, to understand the assembly and fate of endogenous bacteria in rice seeds from the ripening stage to the storage and seedling stages, we employed culture-dependent and metagenomic analyses. Bacterial communities in rice seeds were composed of a few dominant taxa that were introduced at the milky and dough stages, and they persisted during seed maturation. The culturable bacterial population gradually increased during the ripening stage, whereas there was a gradual decrease during storage. Bacteria that persisted during storage proliferated after imbibition and were distributed and established in the shoots and roots of rice seedlings. The storage temperature influenced the abundance of bacteria, which consequently changed the bacterial composition in the shoots and roots of seedlings. Pantoea, Pseudomonas, and Allorhizobium were consistently abundant from seed development to the germination stage. Some endogenous bacterial strains significantly promoted the growth of Arabidopsis and rice plants. Overall, our results indicate that rice seeds are colonized by a few bacterial taxa during seed development, and their relative abundance fluctuates during storage and contributes significantly to the establishment of endophytes in the stems and roots of rice plants. The selected bacterial isolates can be used to improve the growth and health of rice plants. To the best of our knowledge, this is the first study to reveal the dynamics of bacterial populations during storage of rice seeds at different temperatures. The temporal dynamics of the bacterial community during seed storage provide clues for the manipulation of endogenous bacteria in rice plants.

Relationship between bacterial phylotype and specialized metabolite production in the culturable microbiome of two freshwater sponges

Microbial drug discovery programs rely heavily on accessing bacterial diversity from the environment to acquire new specialized metabolite (SM) lead compounds for the therapeutic pipeline. Therefore, knowledge of how commonly culturable bacterial taxa are distributed in nature, in addition to the degree of variation of SM production within those taxa, is critical to informing these front-end discovery efforts and making the overall sample collection and bacterial library creation process more efficient. In the current study, we employed MALDI-TOF mass spectrometry and the bioinformatics pipeline IDBac to analyze diversity within phylotype groupings and SM profiles of hundreds of bacterial isolates from two Eunapius fragilis freshwater sponges, collected 1.5 km apart. We demonstrated that within two sponge samples of the same species, the culturable bacterial populations contained significant overlap in approximate genus-level phylotypes but mostly nonoverlapping populations of isolates when grouped lower than the level of genus. Further, correlations between bacterial phylotype and SM production varied at the species level and below, suggesting SM distribution within bacterial taxa must be analyzed on a case-by-case basis. Our results suggest that two E. fragilis freshwater sponges collected in similar environments can exhibit large culturable diversity on a species-level scale, thus researchers should scrutinize the isolates with analyses that take both phylogeny and SM production into account to optimize the chemical space entering into a downstream bacterial library.

Effects of Nanoformulated Plant Growth Regulator on Culturable Bacterial Population, Microbial Biomass and Enzyme Activities in Two Soil Types

Nanomaterials have many beneficial applications; however, their impacts on the environment necessitate an assessment. Understanding any possible negative effects of nano-based products on soil health needs to be performed before their extensive use in crop production and commercialization. Microorganisms are considered the most sensitive indicators of environmental stresses. Thus, this study assessed the effects of nanoformulated plant growth regulator (HormoGroe®) on the culturable soil bacterial population, dehydrogenase (DHA) and urease (UA) activities, and microbial biomass (MCB) in Lipa clay loam (LCL) and Sariaya sandy loam (SSL) over 75-day incubation period in the laboratory. Results showed the treatments had no significant effect on the culturable bacterial population. HormoGroe® had no adverse impact on DHA in both LCL and SSL. HormoGroe® significantly enhanced UA in SSL at 35 days after amendment (DAA) but significantly decreased UA in LCL at 75 DAA, while it decreased significantly the MCB at 35 and 1 DAA in LCL and SSL, respectively. Regardless of the amendments, LCL had higher DHA and UA but lower MCB than SSL. The effect of HormoGroe® and the non-nanomaterials on the parameters measured was influenced by soil type. In conclusion, HormoGroe® is safe for soil microbiome, mainly bacteria in crop production.

Enrichment of Antibiotic Resistant Bacteria and Antibiotic Resistance Genes by Sulfamethoxazole in the Biological Treatment System of Mariculture Wastewater

Although antibiotics are heavily used in mariculture, only a small portion of the added antibiotics is absorbed. Little is known about the response process of antibiotics, antibiotic resistant bacteria, and antibiotic resistance genes to antibiotic-containing wastewater entering a wastewater treatment system. In this study, an anoxic/aerobic moving bed biofilm reactor (A/O-MBBR) was used to treat marine aquaculture wastewater containing sulfamethoxazole (SMX). The antibiotics and resistance genes in the reactor were then evaluated under selective SMX pressure, and the changing abundance patterns and the response of microbial communities and cultivable resistant bacterial populations were further explored. The results show that with an influent SMX concentration of 500 μg ·L-1 and a hydraulic retention time of 8 h, SMX had a slight effect on the removal rate of NH4+-N and NO2--N, following which the performance gradually recovered. During this stage, SMX removal reached approximately 32%, with more than 78% of SMX removed from the hypoxic zone. The resistance gene was more significantly enriched in the hypoxic zone than in the aerobic zone. In the hypoxic zone, the absolute abundance of gene sul1 increased by 2.43 log, whereas that of gene sul2 increased by 1.71 log. In the region, the absolute abundance of sul1 increased by 1.17 log, whereas that of sul2 increased by 0.91 log. Resistant plate culture and high-throughput sequencing showed that the genus Pseudoalteromonas was the most dominant culturable resistant bacteria in the reactor. The genus Pseudomonas predominated in the uncultured resistant bacteria in the reactor. This study showed that marine aquaculture wastewater containing SMX promotes the enrichment of resistance genes, causing the abundance of some resistant bacteria to increase significantly.

Polydimethylsiloxane-polymethacrylate block copolymers containing quaternary ammonium salts against Fusarium oxysporum f. sp. cubense race 4 in soil: Antifungal activities and pot experiments

Fusarium oxysporum f. sp. cubense race 4 (Foc4) has significant impacts on banana production. Foc4 conidia can survive for many years in the soil, making control difficult. Thus, the inhibition of the conidia that serve as a continual source of inoculum in soil should be an effective way to control banana Fusarium wilt. In this paper, we first investigated the activities of polydimethylsiloxane-polymethacrylate block copolymers containing quaternary ammonium salts (PDMS-b-QPDMAEMA, labeled SixQ5) against Foc4 with microbroth dilution and toxicity plate assays. Then, the environmental behavior of these compounds, i.e., their adsorption and migration in soils, was further studied. Later, the effect and duration of SixQ5 on Foc4 in soil and the effects of SixQ5 on soil microbial diversity were evaluated. In addition, we conducted pot experiments to study the effect of Si5Q5 on banana Fusarium wilt. The results showed that SixQ5 showed a good inhibitory effect on Foc4, was easily adsorbed by the soil and was relatively immobile in soil. The inhibitory effect of SixQ5 on Foc4 was maintained for 30 d under continual inoculation of Foc4 into the soil (Foc4 addition every 2 d), and SixQ5 remained active in the soil for 90 d with Foc4 addition every 10 d. Soil microbial population analysis revealed that the culturable total fungal and bacterial populations increased and the culturable actinobacterial population decreased SixQ5 with addition. Pot experiments showed that Si5Q5 can reduce Foc4 infection in banana seedlings and does not harm banana seedlings. The stability and inhibitory effects on Foc4 demonstrate the potential application of SixQ5 in banana fields to prevent Foc4 conidial infection in the long term.

Lacticaseibacillus casei in Diet of Juvenile Convict Cichlid Fish (Amatitlania nigrofasciata): Evaluating Growth Performance, Digestive Enzyme Activities, Immune Responses, and Stress Resistance.

In this study, growth performance, body composition, digestive enzymes activity, mucosal and immunological parameters, cultivable bacterial populations, and stress resistance were investigated in juvenile convict cichlid fish (Amatitlania nigrofasciata) that received a dietary supplement containing 0 (control), 5×106 (LC1), 5×107 (LC2), and 5×108CFUg-1 diet (LC3) Lacticaseibacillus casei PB-LC39. Two hundred and forty fish (2.44±0.04g) were assigned to twelve experimental aquariums and feed ad libitum three times a day for 8weeks. After final sampling, final body weight, percentage of weight gain (WG %), specific growth rate (SGR), food conversion ratio (FCR), and protein content of whole-body composition were significantly higher (P<0.05) in fish fed LC1 diet than other treatments. Total protease, amylase, and lipase activities were also significantly higher (P<0.05) in fish fed LC1 diet than other groups. Total protein from serum and mucus, lysozyme activity, total immunoglobulin (Ig), and serum globulin were significantly increased (P<0.05) in fish fed LC1 diet compared with other groups. Moreover, total counts of lactic acid bacteria (LAB) in fish gut were significantly higher (P<0.05) by different levels of L. casei PB-LC39 than the control group. Recovery rates of fish fed the probiotic cells, after an air-dive test, was significantly increased (P<0.05) compared with the control group. Therefore, the results showed that L. casei PB-LC39 resulted in improving growth, health status, and stress resistance of fish during the rearing of juvenile convict cichlid fish.

Comparison of the performance of the biofilm sampling methods (swab, sponge, contact agar) in the recovery of Listeria monocytogenes populations considering the seafood environment conditions

AimsThe aim of this study was to evaluate the performance of sampling methods [contact plates, sponges, and swabs] in the recovery of biofilm Listeria monocytogenes populations considering the seafood environment conditions (nature of conditioning, of materials and bacterial species). Methods and resultsDifferent materials (stainless steel, polyvinyl chloride, polyurethane) were conditioned with two fish filtrates, the ready-to-eat the most consumed in Europe (smoked salmon, cod). After, we added the suspension of Listeria monocytogenes, alone or with Pseudomonas fluorescens or Carnobacterium strains, and incubated for 48 h at 8 °C. Then, the 48 h-biofilms were sampled with different methods (contact plates, sponges, and swabs). The cultivable bacterial populations were enumerated on agar, while the L. monocytogenes total and viable populations were quantified by qPCR and propidium monoazide-qPCR (PMA-qPCR), respectively. The amount of L. monocytogenes in biofilms was affected only by the nature of the conditioning with lowest adherent bacteria with cod versus with smoked salmon conditioning. Considering the amount of total population, the swab displayed the lowest values versus the sponges and the contact plates. An explanation was that the observations of the swab by microscopy showed the bacteria trapped within it. The recovery of cultivable bacterial populations was not significantly different with the three sampling methods. On the contrary, we showed that the VBNC populations were only detached by two of three methods (contact plates, sponges) while for the dead populations, those were contact plates and swabs. ConclusionsThe nature of the conditioning influenced the amount of the bacteria in biofilms. And the performance of the recovery of the bacterial populations (dead, VBNC, cultivable) was dependent on the methods used. Significance and impact of the studyThis study showed that the seafood environmental conditions influenced the biofilm formation and the assessment of the efficiency of cleaning and disinfectant operations could be significantly affected by the used sampling methods.

Application of cowdung and sawdust as biostimulants for enhanced bioremediation of diesel contaminated soil

The study was aimed at exploring the use of cow dung (CD) and sawdust (SD) as bio stimulants for enhanced ex-situ bioremediation of diesel fuel contaminated soil. Results of the microbial populations in a gram of soil showed that diesel polluted soil amended with CD had the highest total culturable heterotrophic bacterial (TCHB) population range (1.42 x 106 - 1.56 x 107Cfu/g), total fungal (TF) (1.0 x 106 – 1.60x107Cfu/g), hydrocarbon utilizing bacteria (HUB) (4.5 x 106 – 2.05 x 107Cfu/g) and hydrocarbon utilizing fungi (HUF) (3.5 x 106 – 1.3 x 107Cfu/g) respectively during the study. Diesel polluted soil amended with SD had lower TCHB populations (1.32 x 106 – 2.0 x 107Cfu/g), TF (6.0 x 106 – 3.6 x 107 Cfu/g), HUB (5.4 x 106 – 1.28 x 107Cfu/g) and HUF (1.2 x 106 – 2.0 x 106Cfu/g) respectively, while the microbial populations in option C (control) were TCHB (1.14 x 106 – 1.18 x 107Cfu/g), TF (1.7 x 106 – 7.0 x 106Cfu/g), HUB (4.2 x 106 – 7.8 x 106Cfu/g) and HUF (1.1 x 106 – 5.0 x 107Cfu/g) respectively. Changes in pH values showed reductions in CD (7.10 – 6.20) and SD (5.78 – 4.88) amended soils. Percentage losses in TPH from chromatography results showed diesel polluted soil amended with CD (61.30%), SD (41%) and control (4.11%) respectively. Characterization and identification tests of bacterial isolates revealed that a microbial consortium comprising of the following HUB genera; Bacillus, Pseudomonas, Proteus, Alcaligenes, Klebsiella, Enterobacter, Citrobacter, Corynebacterium and Escherichia coli was implicated in the biodegradation process. Similarly, the HUF encountered were Fusarium, Aspergillus, Microsporium, Penicilium and Mucor. The results suggest that the application of cow dung as biostimulant will enhance the bioremediation of diesel contaminated soil than the application of sawdust.Keywords: Cowdung, Sawdust, Bioremediation, Biostimulation

Effects of bio-organic fertiliser fortified by Bacillus cereus QJ-1 on tobacco bacterial wilt control and soil quality improvement

ABSTRACTIn this study, bio-organic fertiliser (BOF) fortified by Bacillus cereus was used as a biocontrol method to reduce tobacco bacterial wilt disease and improve soil quality. The effects of BOF application on the control of tobacco bacterial wilt disease were compared against conventional fertiliser (CF) and improved fertiliser (IF). Further, comparisons of soil physiochemical and microbiological differences were conducted. BOF was the most effective treatment in controlling tobacco bacterial wilt disease, with a control percentage of 31.43% after 95 d of tobacco transplantation. After BOF treatment, the contents of available nitrogen, available phosphorus and organic matter in rhizosphere soils were all significantly higher than in those of the CF and IF treatments, while available potassium content and soil pH were lower. The BOF treatment significantly promoted the growth of cultivable bacterial populations in the soil compared to the other two treatments, with increases in cells of 178.19% compared against the CF treatment soils. The BOF and IF treatments also significantly promoted the diversity of soil bacteria during the rapid-growth stage compared against the CF treatment. Thus these results suggest that BOF treatment can improve the ecological stability of soils and help mitigate tobacco bacterial wilt disease.

Specific antibacterial activity of copper alloy touch surfaces in five long-term care facilities for older adults

Pathogens involved in healthcare-associated infections can quickly spread in the environment, particularly to frequently touched surfaces, which can be reservoirs for pathogens. The purpose of this study was to investigate naturally occurring bacterial contamination on touch surfaces in five French long-term care facilities and to compare bacterial populations recovered from copper and control surfaces. More than 1300 surfaces were sampled. The collected bacteria were identified to obtain a global view of the cultivable bacterial populations colonizing touch surfaces. Haemolytic colonies and putative pathogens were also screened using specific agar plates and then identified with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. In total, more than 3400 colonies were analysed. Staphylococcus and Micrococcus were the two predominant genera present on touch surfaces, respectively occurring on 51.8% and 48.0% of control surfaces. In these facilities with relatively low bioburden, copper surfaces efficiently reduced the occurrence frequencies of three genera: Staphylococcus, Streptococcus and Roseomonas. Pathogenic species such as Staphylococcus aureus, Enterococcus faecalis and E.faecium were observed in very few samples. In addition, meticillin-resistant S.aureus was observed on five control surfaces and one copper surface. Contamination of healthcare facilities touch surfaces can be the source for the spread of bacteria through the institution. This in situ study shows that the frequency of the contamination as well as the specific bacterial population bioburden is reduced on copper alloy surfaces.

Characterization of the Cultivable Microbiota in Fresh and Stored Mature Human Breast Milk.

Besides nutritional components, breast milk contains diverse microbes, which may be involved in colonization of the infant gut. Expressed milk is often stored for few days in the refrigerator. The aim of this study was to determine the abundance, prevalence and diversity of facultative and strict anaerobic bacteria using culture-dependent and -independent methods, and to determine changes in milk microbial and chemical composition during storage. Samples of mature breast milk from 21 women were collected 3–6 months post-partum and were analyzed fresh or after anaerobic storage for 6 days at 4°C. The cultivable bacterial population was analyzed using the most probable number (MPN) method or plate counts and different media. The abundance of major bacterial groups was determined using quantitative PCR and 16S rRNA gene sequencing. Lactose, lactate, short chain fatty acids (SCFA) and human milk oligosaccharides (HMO) were analyzed using chromatography techniques. Highest mean viable cell counts were obtained in yeast casitone fatty acids (YCFA) broth supplied with mucin (log 4.2 ± 1.8 cells/ml) and lactose (log 3.9 ± 1.4 cells/ml), or Columbia broth (log 3.0 ± 0.7 cells/ml). Mean total bacterial counts estimated by qPCR was 5.3 ± 0.6 log cells/ml, with Firmicutes being the most abundant phylum. The most prevalent bacterial groups were Streptococcus spp. (15/19 samples), Enterobacteriaceae (13/19) and Lactobacillus/Lactococcus/Pediococcus group (12/19). While the average total number of bacterial cells did not change significantly during storage, the prevalence of strict anaerobic Bacteroidetes increased threefold, from 3/19 to 9/19, while in 7 samples Clostridium clusters IV or XIVa became detectable after storage. Major HMO were not degraded. Lactate was present in 18/21 samples after storage (2.3–18.0 mM). Butyrate was detected in 15/21 and 18/21 samples before and after storage, respectively, at concentrations ranging from 2.5 to 5.7 mM. We demonstrate enhanced prevalence and/or abundance of viable strict anaerobes from the Bacteroidetes and Clostridiales after 6-day anaerobic storage of human milk. Our data indicate that anaerobic cold storage did not markedly change total viable bacterial load, while HMO profiles were stable. Anaerobic cold storage of human milk for up to 6 days may be suitable for preserving milk quality for potential microbial transfer to the infant gut.

Synergistic effect of sodium selenite and Pediococcus acidilactici on growth, intestinal bacterial counts, selenium bioavailability, hepatic enzymes and non‐specific immune response in rainbow trout ( Oncorhynchus mykiss )

Lactic acid bacteria (LAB) have a crucial role in inorganic selenium metabolism as well as their known desirable effects on fish. In this study, the synergistic effects of dietary sodium selenite and Pediococcus acidilactici on growth performance, intestinal bacterial counts, selenium bioavailability, hepatic antioxidant enzyme thioredoxin reductase activity and hepatic glycolytic enzyme activity that is hexokinase, phosphofructokinase and pyruvate kinase, and non‐specific immune response such as serum lysozyme and complements C₃, C₄ and ACH₅₀ activity in rainbow trout (Oncorhynchus mykiss) were investigated. Thus, a 3 × 3 factorial design experiment was conducted with nine purified diets including three levels of sodium selenite (0, 1 and 2 mg/kg) and three levels of P. acidilactici (0, 7 and 9 log CFU/g). After 8 weeks of feeding, weight gain and specific growth rate were increased by increasing dietary sodium selenite and P. acidilactici levels compared to control (p .05). Total cultivable bacterial populations after 4 and 8 weeks of the feeding trial were not significantly different among the dietary treatments, while LAB levels were higher in P. acidilactici‐fed groups than in control and selenium‐fed groups (p < .05). Selenocysteine, methylselenocysteine and selenomethionine levels in the intestine of rainbow trout were increased by increasing the sodium selenite and P. acidilactici levels (p < .05), and selenocysteine was found the most selenium species in the trout intestine. The quantity of total selenium in the whole body, intestine, blood, liver and muscle of rainbow trout were increased by increasing the amounts of sodium selenite and P. acidilactici compared to control (p < .05). Hepatic thioredoxin reductase and hexokinase activity were increased by increasing dietary selenium and P. acidilactici levels in comparison to control (p < .05), whereas phosphofructokinase and pyruvate kinase activity in the liver of rainbow trout were not significantly different between the dietary treatments. Serum lysozyme, complements C₃, C₄ and ACH₅₀ activity were enhanced by increasing dietary selenium and P. acidilactici levels compared to control (p < .05). The most synergistic effects of dietary supplements on growth and metabolism of rainbow trout were obtained at 2 mg/kg sodium selenite and 7 log CFU/g P. acidilactici. The findings revealed the synergistic effect of dietary selenium and P. acidilactici on growth and metabolism in rainbow trout (O. mykiss).

Long-term in situ moisture conservation in horti-pasture system improves biological health of degraded land

In situ moisture conservation practices can conserve fertile topsoil and enhance available water in soil profile. We hypothesised that reclaiming degraded land ecologically through tree + pasture + in situ moisture conservation practices would significantly improve soil organic carbon (SOC) and health. Hence, the objectives were a) to identify changes in nutrient cycling enzymes and SOC status due to different in situ soil moisture conservation options in surface and subsurface soil layers, and b) to test the potentiality of soil enzymes to determine long-term nutrient availability. We conducted a long-term experiment involving aonla (Emblica officinalis) trees + pasture (Cenchrus ciliaris + Stylosanthes seabrana) + in situ soil moisture conservation measures viz. staggered contour trenches (T1), continuous contour trenches (T2), stone mulch (T3), vegetative barriers (T4), control (T5) and fallow land (T6) since 2007. Recommended dose of nitrogen (N), phosphorus (P) and potassium (K) were added to all treatments, except T6. SOC concentration increased by ~51 and 31% in T1 and T2, respectively, over T5 in surface (0–15 cm) soil. Culturable bacterial and fungal populations increased by ~20 and 95% in T1 over T5 in surface soil. Activities of all soil enzymes increased in T1 and T2 (ranging from 42 to 289%) over T5 and T6 in both surface and sub-surface (15–30 cm) layers. However, specific activity of phenol oxidase was ~25% lower for T1 than T6, suggesting more efficient SOC sequestration in T1. Moreover, geometric mean enzyme activity of T1 was ~65 and 33% higher than T5 and T3, respectively, in surface soil. Treated soil quality index (T-SQI) of T1 was ~184% higher than T5. Soil functional diversity was also ~1.24 and 1.22 times higher in T1 and T2 than T5, respectively. Peroxidase was the major C degrading enzyme in this ecosystem. Protease, urease and phosphatase significantly influenced N and P availability along with fruit and pasture yields. Importantly, ~96, 62 and 82% variability of SOC, N and P concentrations, respectively, could be attributed to their corresponding enzyme activities. Principal components analysis (PCA) revealed one-way operational role of soil enzymes. Thus, enzymes are potentially important for recycling nutrients from litters, root biomass of fruit trees and grasses to boost their availability in the long run. Adoption of horti-pasture system combined with moisture conservation practices and staggered contour trenches or continuous contour trenches ensured higher above ground biomass yield, SOC, nutrient availability and soil quality. Thus, long-term use of these practices could be recommended for reclamation and improving soil health and crop productivity of degraded lands of central India.

Characterization of the total and viable bacterial and fungal communities associated with the International Space Station surfaces

BackgroundThe International Space Station (ISS) is a closed system inhabited by microorganisms originating from life support systems, cargo, and crew that are exposed to unique selective pressures such as microgravity. To date, mandatory microbial monitoring and observational studies of spacecraft and space stations have been conducted by traditional culture methods, although it is known that many microbes cannot be cultured with standard techniques. To fully appreciate the true number and diversity of microbes that survive in the ISS, molecular and culture-based methods were used to assess microbial communities on ISS surfaces. Samples were taken at eight pre-defined locations during three flight missions spanning 14 months and analyzed upon return to Earth.ResultsThe cultivable bacterial and fungal population ranged from 104 to 109 CFU/m2 depending on location and consisted of various bacterial (Actinobacteria, Firmicutes, and Proteobacteria) and fungal (Ascomycota and Basidiomycota) phyla. Amplicon sequencing detected more bacterial phyla when compared to the culture-based analyses, but both methods identified similar numbers of fungal phyla. Changes in bacterial and fungal load (by culture and qPCR) were observed over time but not across locations. Bacterial community composition changed over time, but not across locations, while fungal community remained the same between samplings and locations. There were no significant differences in community composition and richness after propidium monoazide sample treatment, suggesting that the analyzed DNA was extracted from intact/viable organisms. Moreover, approximately 46% of intact/viable bacteria and 40% of intact/viable fungi could be cultured.ConclusionsThe results reveal a diverse population of bacteria and fungi on ISS environmental surfaces that changed over time but remained similar between locations. The dominant organisms are associated with the human microbiome and may include opportunistic pathogens. This study provides the first comprehensive catalog of both total and intact/viable bacteria and fungi found on surfaces in closed space systems and can be used to help develop safety measures that meet NASA requirements for deep space human habitation. The results of this study can have significant impact on our understanding of other confined built environments on the Earth such as clean rooms used in the pharmaceutical and medical industries.

Screening of plant growth promoting bacteria (PGPB) from rhizosphere and bulk soil of Caragana microphylla in different habitats and their effects on the growth of Arabidopsis seedlings

The rhizospheric bacterial community associated with desert shrub is an important part of sandy land ecosystems. In this study, we isolated and characterized culturable bacteria associated with Caragana microphylla from a wide range of habitats in North China. We chose five habitats with different aridity index values and dune types. In both trials, soil physicochemical parameters and culturable bacterial populations showed the rhizosphere effects. The culturable bacterial communities of rhizosphere and bulk soil were determined using 16s rDNA sequencing. We identified that the proportions of beneficial bacterial strains with plant growth promoting (PGP) traits (nitrogen fixation, phosphate solubilization and indole acetic acid production) were significantly greater in rhizosphere soil than in bulk soil. Some of the strains screened in this study had significant effects on the growth of Arabidopsis seedlings. These effects included inhibiting the elongation of primary roots, promoting lateral root formation and increasing the shoot fresh weight. In conclusion, our study provides a comprehensive survey of the culturable bacterial populations of the rhizosphere in C. microphylla habitats and identifies potential PGPBs that may help develop an efficient revegetation strategy to manage desert land.