Essay On Micro Organisms In Ponds

Environmental description

The characteristics of the surface and bottom water samples from two different feeding types of ponds within five months are summarized in Table 1. As observed, there were no significant differences in the pH values and DO concentrations no matter between surface and bottom or between two types of ponds (p > 0.05). The NH4+-N and NO2-N concentration were also found no significant differences between the surface and bottom from the two type of ponds (p > 0.05). However, both NH4+-N and NO2-N concentrations were found significantly lower in the ponds D than that in the ponds E (p < 0.05). Variations of water temperature, and the NH4+-N, NO2-N, NO3-N, TP, and TOC concentrations of the two types of ponds through five months are shown in Fig. 1. Temperature of the pond water ranged from16.9 ± 0.25 to 32.4 ± 0.06 °C, with the highest record in August. Concentrations of ammonia and nitrite exhibited the same variations in the grass carp fed with sudan grass (D) and those fed with commercial feeds (E). Ammonia and nitrite concentrations were higher in Ponds E than those in Ponds D.

Abundance of bacterial 16S rRNA genes

The abundance of bacterial 16S rRNA genes is shown in Fig. 2 as determined by qPCR from two types of ponds. The abundance of bacterial 16S rRNA genes in all samples had the same changing trend: it showed a decrease first, followed by an increase, a decrease and then an increase again (Fig. 2). The bacterial abundance ranged from3.53 ± 0.62 × 109 to 8.63 ± 1.01 × 1010 copies/mL and from2.81 ± 0.64 × 109 to 7.42 ± 0.87 × 1010 copies/mL for D ponds and E ponds, respectively.

In D ponds, the abundance of bacterial 16S rRNA genes was significantly higher in the surface water layer than in the bottom layer in August (p < 0.05), while no significant differences were observed between them in other months (p > 0.05). Similar results were obtained for E ponds.

The grass carp were fed with sudan grass in July and August in D ponds, and there were no significant differences in the abundance of bacterial 16S rRNA genes between D ponds and E ponds in these two months (p > 0.05). However, in September when the feed was changed to commercial feed again, the abundance of bacterial 16S rRNA genes in D ponds was significantly lower than that of E ponds (p < 0.05), and no differences were observed between the two types of ponds in October with further supplement of commercial feed (p > 0.05).

Illumina MiSeq sequencing results

After the removal of unqualified reads, the Illumina MiSeq sequencing analysis of fifty water samples yielded 745,337 sequences, with an average length of about 396 bp in the V4–V5 hypervariable regions of the 16S rRNA gene. As summarized in Table 2, the number of operational taxonomy units (OTUs) in the E ponds was larger than that in D ponds no matter in the surface or bottom. By contrast, the Shannon indices showed higher bacterial diversity in D ponds than those in E ponds both on the surface and bottom. It was also noted that bacterial diversity in D ponds was greater than that in E ponds at the bottom layer in August. The rarefaction curves of the twenty samples at the 3% distance cutoff level revealed that the bacterial phylotype richness of sample 7Es was considerably higher than that of other samples (Fig. 3).

Bacterial community composition

RDP Classifier was used to assign the effective sequence tags into different phylogenetic bacterial taxa. Figure 4 shows the relative abundance of bacterial community at the level of phylum. Proteobacteria, Cyanobacteria, Bacteroidetes and Actinobacteria were four phyla abundant in all the samples from the two types of ponds. Except for July, Proteobacteria were the most abundant phylum in all experimental months, accounting for 34.95% of the total effective bacterial sequences, followed by Cyanobacteria (averaging at 18.8%), Bacteroidetes (averaging at 15.8%) and Actinobacteria (averaging at 14.7%). Other major phyla (average abundance  > 1%) of the bacterial communities of the twenty samples included Firmicutes (4.6%), Planctomycetes (3.5%), Chloroflexi (1.9%) and Chlorobi (1.2%). Proteobacteria, Bacteroidetes, and Actinobacteria had the same varying tendency during the five months at the four sampling sites. However, in the surface water layer of D ponds, Cyanobacteria were first increased and then decreased during August to October, while in other three sampling sites it had the opposite varying tendency.

To further understand the differences in dominant phyla between D and E ponds, the mean relative abundance of the four dominant OTUs at each sampling site in the five months was analyzed (Supplementary Fig. S1). Proteobacteria were found to be the most dominant phylum in all samples (except for the samples in July) followed by Actinobacteria, Bacteroidetes and Cyanobacteria. As observed, the relative abundance of Proteobacteria and Actinobacteria in D ponds were both higher than those in E ponds. However, the relative abundance of Bacteroidetes in D ponds was lower than that in E ponds. The relative abundance of Cyanobacteria fluctuated in D and E ponds, and showed no particular trends. For the rest, Firmicutes and Chlorobi were more abundant in E ponds than that in D ponds (4.0% and 1.1%, respectively), whereas Chloroflexi and Planctomycetes were less abundant.

To conduct a more detailed analysis of the composition of the communities in the water layers, all the reads had been assigned to a phylum into classes (Fig. 5). As observed, Betaproteobacteria were the dominant class among Proteobacteria in all water samples, accounting for 15.7%, followed by Alphaproteobacteria, Gammaproteobacteria and Deltaproteobacteria. Among these classes, Gammaproteobacteria were more abundant in D ponds (9.4% in surface layer, 8.5% in bottom layer) than in E ponds (5.9% in surface layer, 7.6% in bottom layer). However, the mean relative abundance of the five subdivisions within Proteobacteria in the two types of ponds was not significantly different over the five months (p  >  0.05). Interestingly, in D ponds, Epsilonproteobacteria were only detected in June and July, averaging at 0.006% in total effective sequences, whereas in E ponds, they were detected in June, July and August, averaging at 0.003%. In addition, Deferribacteres and Thermotogae were only detected in the surface water of E ponds in June.

Hierarchically clustered heatmap showed the similarities and differences of these twenty bacterial communities at genus level (Fig. 6). Cluster analysis classified the samples into five clusters. Except for the samples from September, other samples from the same types of the pond were grouped together first, and then the samples in the same month from different types of the pond were clustered together.

At genus level, some interesting differences between the two types of ponds during the five months were observed. Some probiotics, such as Comamonadaceae unclassified and Bacillales unclassified were found to be significantly higher in D ponds than those in E ponds (p < 0.05) in July and August when the feed was changed to sudan grass feed. On the other hand, some potential pathogens such as Acinetobacter and Aeromonas were found to be significantly lower in D ponds than those in E ponds (p < 0.05) in August.

Similarity analysis of the twenty water samples

The weighted UniFrac clustering method was used to calculate the similarity or dissimilarity of the obtained sequences among different samples25. As shown in Fig. 7, based on abundances of orders, the bacterial communities in the twenty samples could be clustered into five groups, which included all four samples from one of the five months respectively: Group I (September), Group II (August), Group III (October), Group IV (July), Group V (June). Except for those from the samples in September, the communities from the same type of ponds were clustered together.

PCoA was used to estimate the similarities among different water samples using three different approaches: RDP Classifier taxa, OTUs and UniFrac, and PCoA (Fig. 8). The first principal coordinate of the weighted analysis accounted for 47.19% of the variation in the data. It clearly separated the samples in June from those in other four months. PC2 accounted for 20.79% of the variance in the bacterial communities.

Microbial community composition in relation to environmental variables

CCA was used to establish the relationships between the environmental factors and the bacterial community (Fig. 9). CCA plot was carried out using OUTs data together with environmental data (ammonia, nitrite, nitrate, total phosphorus, total organic carbon, temperature, pH, and dissolved oxygen). According to Monte Carlo permutation test (499 permutations), the significant relationships between environmental variables and canonical axes were analyzed by using Canoco program.

Based on the 5% level in a partial Monte Carlo permutation test, the bacterial community and structure were significantly linked (p <  0.05) to the water environment factors. As shown in Fig. 9, the water samples were clearly clustered according to sampling time rather than sampling water layer. CCA results explained 36.8% and 18.9% of the variation in the first two axes, respectively (Fig. 9). Dissolved oxygen, temperature and total organic carbon were the most important environmental factors to influence the water community composition, and were positively correlated with Axis 1 (p < 0.05) (Table 3). Axis 2 had a positive correlation with temperature, nitrite and nitrate (p < 0.05), but was negatively correlated with ammonia (p < 0.01) (Table 3), suggesting that Axis 2 had a gradient in temperature, nitrite, nitrate and ammonia. By contrast, other nutrient factors (pH and total phosphorus) had no significant correlation to bacterial community (p > 0.05).

7.1. The Rationale of the Use of Fecal Indicator Bacteria

The most important bacterial gastrointestinal diseases transmitted through water are cholera, salmonellosis and shigellosis. These diseases are mainly transmitted through water (and food) contaminated with feces of patients. Drinking water can be contaminated with these pathogenic bacteria, and this is an issue of great concern. However, the presence of pathogenic bacteria in water is sporadic and erratic, levels are low, and the isolation and culture of these bacteria is not straightforward. For these reasons, routine water microbiological analysis does not include the detection of pathogenic bacteria. However, safe water demands that water is free from pathogenic bacteria [57].

The conciliation of the two needs was met by the discovery and testing of indicator bacteria. Water contaminated with pathogenic species also has the normal inhabitants of the human intestine. A good bacterial indicator of fecal pollution should fulfill the following criteria: (1) exist in high numbers in the human intestine and feces; (2) not be pathogenic to humans; (3) easily, reliably and cheaply detectable in environmental waters. Additionally, the following requisites should be met if possible: (4) does not multiply outside the enteric environment; (5) in environmental waters, the indicator should exist in greater numbers than eventual pathogenic bacteria; (6) the indicators should have a similar die-off behavior as the pathogens; (7) if human fecal pollution is to be separated from animal pollution, the indicator should not be very common in the intestine of farm and domestic animals [1,4,6,57,58]. The usefulness of indicator bacteria in predicting the presence of pathogens was well illustrated in many studies, namely by Wilkes et al. [59].

7.2. The Composition of Human and Animal Feces

Microbiological analysis of the human feces was important in order to structure and validate the use of fecal indicator bacteria in environmental waters. Bacteria present in feces are naturally derived from the microbiota of the human gastrointestinal tract.

Although bacteria are distributed throughout the human gastrointestinal tract, the major concentration of microbes and metabolic activity can be found in the large intestine. The upper bowel (stomach, duodenum, and jejunum) has a sparse microbiota with up to 105 CFU/ml of contents. From the ileum on, bacterial concentrations gradually increase reaching in the colon 1010 to 1011 CFU/g [60].

It has been estimated that at least 500–1,000 different microbial species exist in the human gastrointestinal microbiota, although on a quantitative basis 10–20 genera usually predominate (Table 6). The total number of microbial genes in the human gastrointestinal tract has been estimated as 2–4 million. This represents an enormous metabolic potential which is far greater than that possessed by the human host [60,64].

Table 6.

Total viable count in feces of healthy humans (children, adults and elderly)a.

The composition of feces from an individual is stable at genus level, but the species composition can vary markedly from day to day. The relative proportion of intestinal bacterial groups can vary between individuals [60,64].

The microflora of the human gastrointestinal tract is dominated by obligate anaerobes, which are ca. 103 more abundant than facultative anaerobes. The main anaerobic genera are Bacteroides, Eubacterium and Bifidobacteria. These organisms account for ca. 90% of the cultivable human fecal bacteria. Bacteroides (mainly B. thetaiotaomicron and B. vulgatus) are the most abundant organism in the human feces and account for 20–30% of cultivable bacteria. The most abundant facultative anaerobes are Enterococci and Enterobacteriaceae. The main Enterobacteriaceae genera are Escherichia, Citrobacter, Klebsiella, Proteus and Enterobacter. Citrobacter and Klebsiella are present in most individuals although in low numbers. Proteus and Enterobacter are only present in a minority of humans [64].

A variety of molecular techniques have been used to study the microbial composition of the human gastrointestinal tract. Results yielded by these studies have shown that many microbes detected by molecular techniques are not isolable by conventional culture-based methods. The presence of high proportions of bifidobacteria detected by culture-based methods is not supported by the results of molecular-based studies. However, the results of molecular-based approaches support many of the findings derived from culture-based methods: the dominance of the obligate anaerobes over facultative anaerobes; the presence of high counts of Bacteroides, Clostridium and Eubacterium [64].

Anaerobic bacteria such as Bacteroides and Eubacterium are not easily cultured by conventional techniques since require incubation chambers with nitrogen atmosphere. Bifidobacterium and Lactobacillus tolerate some oxygen but are fastidious bacteria growing very slowly in culture media. Therefore, these four genera are not adequate to be used as indicators of fecal pollution (the introduction of molecular techniques may improve the situation). Citrobacter, Klebsiella and Enterobacter are present in low numbers in the human intestine and are widespread in environmental waters, and therefore are also not suitable as indicators of fecal pollution. Clostridium, Streptococcus and Escherichia do not suffer from these drawbacks. Therefore, their suitability as fecal indicators has been tested since several decades.

7.3. Fecal Bacteria in Their Hosts and in the Environment

7.3.1. Bacteroides

The traditional genus Bacteroides included Gram-negative, non-sporeforming, anaerobic pleiomorphic rods. Many species have been transferred to other genera—Mitsuokella, Porphyromonas, Prevotella, Ruminobacter. Bacteroides are the most abundant bacteria in human feces. In animal feces, on the contrary, Bacteroides are present at low numbers. Although anaerobic, Bacteroides are among the most tolerant to oxygen of all anaerobic human gastrointestinal species. B. thetaiotaomicron is one of the most abundant species in the lower regions of the human gastrointestinal tract. Bacteroides have a high pathogenic potential and account for approximately two-thirds of all anaerobes isolated from clinical specimens. The most frequently isolated species has been B. fragilis. The survival of Bacteroides in environmental waters is usually much lower than the survival of coliforms [64,65].

7.3.2. Eubacterium

The traditional genus Eubacterium included anaerobic non-sporeforming Gram-positive rods. Some species have been transferred to other genera—Actinobaculum, Atopobium, Collinsella, Dorea, Eggerthella, Mogibacterium, Pseudoramibacter and Slackia. Cells are not very aerotolerant. Species isolated from the human gastrointestinal tract include: E. barkeri, E. biforme, E. contortum, E. cylindrioides, E. hadrum, E. limosum, E. moniliforme, E. rectal and E. ventricosum [64].

7.3.3. Bifidobacterium

Bifidobacteria are Gram-positive, non-sporeforming, pleiomorphic rods. Bifidobacteria are anaerobic (some species tolerate oxygen in the presence of carbon dioxide) or facultative anaerobic. The optimum growth temperature is 35–39 °C. The genus Bifidobacterium contains ca. 25 species, most of which have been detected in the human gastrointestinal tract [64–66].

Bifidobacteria are present in high numbers in the feces of humans and some animals. Several Bifidobacterium species are specific either for humans or for animals. B. cuniculi and B. magnum have only been found in rabbit fecal samples, B. gallinarum and B. pullorum only in the intestine of chickens and B. suis only in piglet feces. In human feces, the species composition changes with the age of the individual. In the intestine of infants B. breve and B. longum generally predominate. In the adult, B. adolescentis, B. catenulatum, B. pseudocatenulatum and B. longum are the dominant species. In both human and animal feces, bifibobacteria are always much more abundant than coliforms [64–66].

Bifidobacteria have been found in sewage and polluted environmental waters, but appears to be absent from unpolluted or pristine environments such as springs and unpolluted soil. This results from the fact that upon introduction into the environment, bifidobacteria decrease appreciably in numbers, probably due to their stringent growth requirements. Bifidobacteria grow poorly below 30 °C and have rigorous nutrient requirements. Reports on the survival of bifidobacteria in environmental waters indicate that their survival is lower than that of coliforms [64–66].

The presence of bifidobacteria in the environment is therefore considered an indicator of fecal contamination. Since some species are specific for humans and animals, the identification of Bifidobacterium species present in the polluted water could, in principle, provide information on the origin of fecal pollution [64–66].

A study carried out in a highly contaminated stream near Bologna, Italy, revealed that B. adolescentis, B. catenulatum, B. longum, B. pseudocatenulatum and B. thermophilum were the most representative species, whereas B. angulatum, B. animalis subsp. animalis (B. animalis), B. breve, B. choerinum, B. minimum, B. pseudolongum subsp. globosum (B. globosum) and B. subtile occurred only in low numbers [66].

Bifidobacteria are the less studied of all fecal bacteria, due to the technical difficulties in their isolation and cultivation. Other Gram-positive bacteria, such as Streptococcus and Lactobacillus, which may occur in higher numbers than bifidobacteria, can inhibit their growth. Although selective media has been designed for the isolation of bifidobacteria from environmental waters, the outcome is still unsatisfactory, with appreciable numbers of false positives and low recovery percentages [64–66].

7.3.4. Clostridia

The genus Clostridium is one of the largest genera of the prokaryotes containing 168 validly published species. From these, 77 (including C. perfringens) are considered to belong to a united group—Clostridium sensu stricto [64,67,68].

Clostridia are Gram-positive rods, forming endospores. Most of the clostridial species are motile with peritrichous flagellation. Cells are catalase-negative and do not carry out a dissimilatory sulphate reduction. Clostridia usually produce mixtures of organic acids and alcohols from carbohydrates and proteins. Many species are saccharolytic and proteolytic. Some species fix atmospheric dinitrogen [64,67,68].

The genus Clostridium includes psychrophilic, mesophilic, and thermophilic species. The major role of these organisms in nature is in the degradation of organic material to acids, alcohols, CO2, H2, and minerals. Frequently, a butyric acid smell is associated with the proliferation of clostridia. The ability to form spores that resist dryness, heat, and aerobic conditions makes the clostridia ubiquitous [64,67,68].

Most species are obligate anaerobic, although tolerance to oxygen occurs. Oxygen sensitivity restricts the habitat of the clostridia to anaerobic areas or areas with low oxygen tensions. Growing and dividing clostridia will, therefore, not be found in air saturated surface layers of lakes and rivers or on the surface of organic material and soil. Clostridial spores, however, are present with high probability in these environments, and will germinate when oxygen is exhausted and when appropriate nutrients are present [64,67,68].

C. perfringens ferment lactose, sucrose and inositol with the production of gas, produce a stormy clot fermentation with milk, reduce nitrate, hydrolyze gelatin and produce lecithinase and acid phosphatase. The species is divided into five types, A to E, on the basis of production of major lethal toxins [68,69].

C. perfringens appears to be a universal component of the human and animal intestine, since has been isolated from the intestinal contents of every animal that has been studied. Humans carry C. perfringens as part of the normal endogenous flora. The main site of carriage is the distal gastrointestinal tract. The principal habitats of type A are the soil and the intestines of humans, animals, and birds. Types B, C, D, and E appears to be obligate parasites of animals and occasionally are found in humans [68,69].

Clostridium perfringens is the most frequently isolated Clostridium in clinical microbiology laboratories, although it seldom causes serious infections. C. perfringens is isolated from infections in humans and the organism most commonly found in gas gangrene in humans. C. perfringens is most commonly isolated from infections derived from the colonic flora, namely peritonitis or abdominal abscess [68,69].

This organism is a common cause of food poisoning due to the formation of the enterotoxin in the intestine. C. perfringens food poisoning is seldom fatal, being marked by diarrhea and nausea, with no vomiting and no fever [68,69].

Sources yielding C. perfringens include soil and marine sediment samples worldwide, clothing, raw milk, cheese, semi-preserved meat products, and venison. Like E. coli, C. perfringens does not multiply in most water environments and is a highly specific indicator of fecal pollution. Berzirtzoglou et al. [70] reported a comparative study on the occurrence of vegetative cells and spores of Clostridium perfringens in a polluted station of the lake Pamvotis, in rural North-West Greece. The numbers of C. perfringens varied according to the water depth. Sporulated forms were found in all sampling sites with the exception of the surface sampling.

7.3.5. Lactobacillus

Lactobacilli are non-sporeforming Gram-positive long rods. There are more than thirty species in the genus. Most are microaerophillic, although some are obligate anaerobes. Cells are catalase-negative and obtain their energy by the fermentation of sugars, producing a variety of acids, alcohol and carbon dioxide. Lactobacilli have complex nutritional requirements and in agarized media may need the supplementation with aminoacids, peptides, fatty-acid esters, salts, nucleic acid derivatives and vitamins. Lactobacilli very rarely cause infections in humans [64].

7.3.6. Enterococci

Enterococci are Gram-positive, non-sporeforming, catalase-negative ovoid cells. Cells occur singly, in pairs or short chains. Optimal growth for most species is 35–37 °C. Some will grow at 42–45 °C and at 10 °C. Growth requires complex nutrients but is usually abundant on commonly used bacteriological media. Cells are resistant to 40% bile, 0.4% azide, 6.5% sodium chloride, have β-glucosidase and hydrolyze esculin. The enterococci are facultative anaerobic but prefer anaerobic conditions [64,71].

The genus was separated from Streptococcus in the 1980s. Enterococci form relatively distinct groups. Members of such groups exhibit similar phenotypic characteristics and species delimitation can be difficult. The E. faecalis group contains, among others, E. faecalis. The E. avium group contains, among others, E. avium. The E. faecium group contains, among others, E. faecium, E. durans and E. hirae. The E. gallinarum group contains, among others, E. gallinarum [64

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