Molecular Biological Methods in

Environmental Engineering
Guocai Zhang1, Li Wei2,5*, Chein-Chi Chang3,4*,Yuhua Zhang5, Dong Wei2
5Guangzhou
ABSTRACT: Bacteria, acting as catalysts, perform the HKUST Fok Ying Tung Research
Institute ,Guang Zhou, China
function of degrading pollutants. Molecular biological

techniques play an important role in research on the

of important advancements in molecular techniques and
community analysis, the composition and the functions of
highlight the application of these methods in diverse
complex microbial communities. The development of
environments.
secondary high-throughput pyrosequencing techiniques

enhances the understanding of the composition of the

microbial community. The literatures of 2015 indicated KEYWORDS: Microbial community and function,

that 16S rDNA gene as genetic tag is still the important microbial metagenomics, microbial transcriptome,

method for bacteria identification and classification. 454 microbial protein, microbial metabolomics.

high doi: 10.2175/106143016X14696400494371

throughput sequencing and Illumina MiSeq sequencing

have been the primary and widely recognized methods to Bacteria identification and classification based on 16S

analyze the microbial. This review will provide rDNA

environmental engineers and microbiologists an overview 16S rDNA is regarded as the most useful and

_______________________________________________ common molecular clock in research on bacteria

1College identification and classification. The characteristics of 16S

of Forestry, Northeast Forestry University,
Harbin, 150040, China
rDNA are small variety, large content (i.e., approximatly
2*State Key Laboratory of Urban Water Resource and
80% of bacterial RNA), modest molecular size and
Environment, Harbin Institute of Technology,
Harbin,Heilongjiang Province, China,*Corresponding present in all organisms. The molecular size of 16S rDNA
author, Email: weilihit@126.com
is appoximately 1.5 Kb, which enables reflection of
3Department of Environmental Science and Engineering,
differences among various species. Moreover, the
School of Energy and Power Engineering, Inner Mongolia
sequence of 16S rDNA can be obtained easily through the
University of Technology, Hohhot 010051, China
application of sequencing techniques. Therefore 16S
*Corresponding author, Email: chang87@gmail.com
rDNA receive high acceptance among bacteriologists and
4Department of Engineering and Technical Services, DC
taxonomists.
Water and Sewer Authority, Washington, DC 20032, USA

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
 The ability of the MinIONTM nanopore Wang et al. (2015) isolated the bacteria from

sequencer was tested by Kilianski et al. (2015). After luminescent-infected shrimps. The pathogen, a

running 6 hours, sufficient data were generated to identify luminescent bacterium named VH1 was identified as

an E.coli sample down to the species level from 16S Vibrio campbellii based on MLSA analysis (16S rDNA,

rDNA amplicons. Three poxviruses (cowpox, vaccinia- rpoD and toxR).

MVA, and vaccinia-Lister) were identified and Lagier et al. (2015) isolated Alistipes timonensis

differentiated down to the strain level, despite over 98% strain JC136T sp. nov. from the fecal flora of a healthy

identity between the vaccinia strains. patient, which is an obligate anaerobic rod. A.timonensis

Parlapani et al. (2015) conducted an exploration is the type strain of A. Timonensis sp. nov., a new species

of initial and spoilage microbiota of sea bream fillets by within the genus Alistipes. The analysis showed that

16S rRNA gene sequence analysis of isolates grown on 3,497,779 bp long genome (one chromosome but no

plates. Results showed that initial microbiota were plasmid) contained 2,742 protein-coding and 50 RNA

dominated by Pseudomonas fluorescens, Psychrobacter genes, including three rRNA genes.

and Macrococcus caseolyticus. At the end of shelf life, Buffiie et al. determined that Clostridium scindens, a

different phylotypes of Pseudomonas were found to bile acid 7ɑ-dehydroxylating intestinal bacterium, is

dominate in most cases, while Pseudomonas veronii associated with resistance to C.difficile infection based on

dominated in fillets under modified atmosphere packaging resistance-associated OTUs they identified and, upon

(MAP) at 0℃. Moreover, in fillet under map at 5℃, new administration, enhances resistance to infection in a

dominant species such as Carnobacterium secondary bile acid dependent fashion. They also

maltaromaticum, Carnobacterium divergens and identified a probiotic candidate that corrects a clinically

Vagococcus fluvialils were revealed. relevant microbiome deficiency. These finding have

Seruga et al. (2015) firstly identified implications for the rational design of targeted

phytoplasmas from the ribosomal subgroup 16SrXII-A antimicrobials as well as microbiome-based diagnostics

(stolbur) by PCR and RFLP analyses of 16S rRNA gene and therapeutics for individuals at risk of C.difficile

and elongation factor-Tu phytoplasma gene (tuf) in infection.

symptomatic grapevines. It was found that amplification Sanchez et al. (2015) isolated three strains of

and RFLP results of 16S rDNA were not influenced by the sulfate-reducing bacteria (M1T,D, and E) from acidic

method used, but RFLP patterns of tuf gene revealed sediments and they were characterized phylogenetically

additional bands if a less time consuming method was and physiologically. Cloning of the 16S rRNA gene was

applied. performed to determine the phylogenetic affiliation of the

isolatees, which confirmed that they were affiliated to the

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
genus Desulfosporosinus of the phylum Firmicutes. Cell accelerate the development of microbial community

morphology, motility, and spore formation were examined analysis.

by phase contrast microscopy using a Leica DM2000 Llado et al. (2015) assessed soil microbial

microscope. community through classical (Most Probably Number -

Allen-Daniels et al. (2015) sequenced the entire MPN) and molecular tools (denaturing gradient gel

genomes of 2 amniotic fluid isolates and a placental electrophoresis-DGGE and pyrosequencing).

isolate of M. hominis from pregnancies. They identified 2 Pyrosequencing data revealed that Alpha and

genes that encode surface-located membrane proteins in Gammaproteobacteria were the most abundant bacterial

the sequenced amniotic fluid/placental isolates that were groups whereas Fusarium and Scedosporium were the

truncated severely in PG21. They also identified, for the main fungal genera in the initial contaminated soil. After

first time, a microbial gene of unknown function that is bioremediation assays, it was foundt that surfactant Brij

referred to in this study as gene of interest C that was 30 hampers 4-ring PAHs degradation due to toxicity over

associated significantly with bacterial burden in amniotic Actinobacteria and Bacteroidetes populations. A high

fluid and the risk of preterm delivery in patients with prevalence of Fusarium and Scedosporium population was

preterm labor. revealed during soil bioremediation.

Basak et al. (2015) assessed the influence of

Application of microbial community analysis in temporal and spatial variation on the microbial

environmental treatment community composition in the unique coastal mangrove

The application of microorganisms in of Sundarbans using parallel 16S rRNA gene

environmental management, and various types of pyrosequencing. 117 Mbp of data was obtained in the total

bioreactors for wastewater, solid waste and waste gas sediment DNA. The taxonimic analysis of the

treatment, are based on bacteria, which constructs a pyrosequencing data was grouped into 24 different phyla.

complex microbial system. Different pollutants, treatment Proteobacteria were the most dominat phyla with

processes and influencing factors play key roles in predominance of Deltaproteobacteria,

construction of microbial system. Nowadays, the Alphaproteobacteria, and Gammaproteobacteria within

emphasis of the research are the composition of microbial the sediment. Besides, the following major phyla were

community, especially the analysis of the composition of detected: Actinobacteria, Bacteroidetes, Planctomycetes,

dominant microorganisms, including the distribution, Acidobacteria, Chloroflexi, Cyanobacteriam, Nitrospira,

succession and spatial variation of those microorganisms. and Firmicutes. Further taxonomic analysis revealed

Secondary high-throughput sequence techniques abundance of micro-aerophilic and anaerobic microbial

population in the surface layers.

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 Chaillou et al. (2015) conducted 16S rRNA 16S rRNA data. Firmicutes and Proteobacteria accounted

gene pyrosequencing on 160 samples of fresh and spoiled for appoximately 57% of the microbiome. Around 70% of

food to comparatively explore the bacterial communities the detected thermophiles were strict anaerobes; however,

associated with four meat products and four seafood Hydrogenobacter spp., obligate chemolithotrophic

products. It was found that fresh products were thermophiles, represented one of the major taxa. Several

contaminated in part by a microbiota similar to that found thermophilic photosynthetic microorganisms and

on the skin and in the fur of animals. The operational aciothermophiles were also detected.

taxonomic unit (OTU) assemblage associated with Etyemez et al. (2015) determined the diversity

spoilage was shaped by low storage temperature, and composition of bacterial communities within the

packaging and the nutritional value of the food matrix intestinal ecosystem of farmed rainbow trout. In this

itself. Moreover, putative new clades of dominant, study, genomic DNA was isolated from the intestinal

previously undescribed bacteria occurring on spoiled mucus and combined into groups of four samples each for

seafood was identified. pyrosequencing analysis of bacterial 16S rRNA genes.

Portune et al. (2015) applied 16S rDNA tag The results showed ten genera affiliated to the OTUs, four

pyrosequencing to styrene-degrading biofilters. Results most abundant genera and six to a lesser extent.

showed that bacterial genera Azoarcus and Pseudomonas Zhang, Huo et al. (2015) investigated the use of

were among the dominant classified genera in the pyrosequencing for evaluating the impacts of nutrition on

biofilter. Canonical correspondence analysis (CCA) and changes in the composition of milk microbiota. High-

correlation analysis revealed that the genera concentrated feeding may increase the risk of dairy cows

Brevundimonas, Hydrogenophaga, and Achroombacter suffering from mastitis, due to the higher proportion of

may play important roles in styrene degradation under some mastitis-causing pathogen bacteria, such as

increasing styrene concentrations. Fluorescence in situ Stenotrophomonas maltophilia, Streptococcus parauberis

hybridization (FISH) was used to compare to results from and Brevundim onas diminuta, as well as of

pyrosequencing, and showed discrepancies between the psychrotrophic bacteria, such as Pseudomonas,

two methods in the detected percentages of numerous Brevundimonas, Sphingobacterium, Alcaligenes,

taxonimic groups. Enterobacter and Lactobacillus. It may also decrease the

Chan, Chan et al. (2015) collected a sample organoleptic quality of raw milk and dairy products, and

from the middle of the stream in the Sungai Klah hot limit the shelf life of processed fluid milk.

spring. The sample was analyzed at the 16S rRNA V3-V4 Xiong, Zhao et al. (2015) assessed soil bacterial

region by amplicon metagenome sequencing. Results and fungal communities across vanilla continuous

showed that over 35 phyla were detected by analyzing the cropping time-series fields through deep pyrosequencing

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of 16S rRNA genes and internal transcribed spacer (ITS) cultivation studies and DNA stable isotope probing
13C-monomethylamine
regions. The results showed that soil fungal diversity (DNA-SIP) using (MMA). The

index increased with consecutive cropping years, whereas SIP experiments revealed that the obligate methylotroph

soil bacterial diversity was relatively stable. Moreover, the Methylotenera mobilis is one of the dominant MMA

alteration of the soil microbial community membership utilisers in the cave. The DNA-SIP experiments also

and structure, due to long-term continuous cropping showed that a new facultative methylotroph isolated,

would lead to soil weakness and vanilla stem wilt disease. Catellibacterium sp. LW-1 might probably be one of the

Huang et al. (2015) investigated phylogenetic most active MMA utilisers in Movile Cave. Methylated

and functional bacterial communities for elemental sulfur amines were also used as a nitrogen source by a wide

recovery and nitrate removal. Results showed that S2- range of non-methylotrophic bacteria in cave.

/NO3- (S/N) molar ratio of 5/6 was optimized for S0 16S rRNA libraries. Otlewska et al. (2015)

recovery and nitrate removal, and also obtained used the construction of 16S rRNA coline libraries to

predominant desulfurization and denitrification genera. analyze the bacterial communities in four samples of

Bacterial community and genetic activity remarkable historical materials (plaster, brick and wood). In total, 138

modified as S/N ratio altered. Under lower S/N ratios, partial 16S rRNA gene sequences (~600bp) were obtained

autotrophic S2- oxidization genera dominated and and compared. The clones belonged to phyla

functioned, while under higher S/N ratios, NO3- reduction Proteobacteria, Actinobacteria, Firmicutes, and

and S0 over oxidization genera functioned. Bacteroidetes. The plaster samples predominatly

Zhang, Cai et al. (2015) investigated the impacts contained clones closely related to Actinobacteria and

of aeration strategies on the evolution of dissolved organic Alphaproteobacteria, brick samples contained

matter (DOM) and the microbial community in sludge Gammaproteobacteria, while wood samples had

biodrying. Results showed that higher temperature Actinobacteria clones.

accelerated the pile to enter the DOM and microbiology Kostovcik et al. (2015) used multiplexed

stable stage. After degradation of easily degradable pyrosequencing of amplified internal transcribed spacer II

components in the DOM primarily in the thermophilic (ITS2) ribosomal DNA (rDNA) libraries to document the

phase, the DOM components changed, which resulted in communities of fungal associates and symbionts inside the

the microbial community changing considerably in the mycangia (fungus transfer organ) of three ambrosia beetle

following mesophilic stage. species, Xyleborus affinis, Xyleborus ferrugineus and

Wischer et al. (2015) investigated the role of Xylosandrus crassiusculus. Ninty-three beetle samples

methylated amines as a carbon and nitrogen source for were processed. It was reported that the composition of

bacteria in Movile Cave using a combination of fungal assemblages was significantly correlated with

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
beetle species but not with locality. The type of the rice fields were irrigated with river water

mycangium determined specificity. Overall, the initial contaminated by Fe-S-rich acid mine drainage.

analysis of three beetle species suggests that each clade of Li, Dai et al. (2015) firstly used biodrying for

ambrosia beetles and each mycangium type may support a postpreatment of anaerobically digested sludge (ADS).

functionally and taxonomically distinct symbiosis. Wheat residues was used as bulking agents to improve the

High-throughput pyrosequencing. Wilkins et ADS quality and reduce its amount. Thermal balance

al. (2015) examined the archaeal communities from three analysis showed the contribution of the substrates to the

sludge digesters and one wastewater digester using high- heat. Also, 454 pyrosequencing technique implied the

throughput pyrosequencing of the methyl coenzyme M obvious succession from the anaerobic to aerobic

reductase (mcrA) and 16S rRNA genes. Results indicated microorganisms during the process. Results indicated that

that hydrogenotrophic and acetoclastic methanogenesis the biodegradation of bulking agent played a key role in

was occurring, due to the detection of Methanobacterials, the ADS biodrying process, with which the ADS

Methanomicrobiales, and Methanosarcinales in each biodrying gained the desired performance. Some

digester. Taxonimic classification of the mcrA genes dominant Firmicutes seemed to relate with organic matter

suggested that these digesters were dominated by degradation of the substrates.

acetoclastic methanogens, particularly Methanosarcinales. Liu et al. (2015) applied 454 pyrosequencing for

Moreover, a significant positive correlation between culture-independent assessments of the bacterial

methane production and the abundance of mcrA community composition in a swine wastewater anaerobic

transcripts in digester, supporting the mcrA gene as a reactor. Results indicated that the community contained

biomarker for methane yield. >2500 OTUs and was dominated by three phyla:

Sun et al. (2015) selected five rice paddies for Bacteroidetes, Firmicutes, and Proteobacteria. A

geochemical and microbial community analysis. simulated organic shock induced dynamic changes in

Microbial communities were characterized by high- community composition, which was re-stabilized after

throughput sequencing, which showed 39 different approximately a threefold hydraulic retention time (HRT).

phyla/groups in these samples. Canonical correspondence After restabilization, the community composition became

analysis suggested that pH, sulfate, and nitrate were vital similar to that observed before the shock, rather than

factors that shaped the microbial community structure. reaching a new equilibrium.

According to the results, Proteobacteria was the most Shu et al. (2015) investigated microbial

abundant phylum in all samples. Moreover, a wide communities of anaerobic sludge by 454 pyrosequencing

diversity of Fe- and S-related bacteria were detected, as and qPCR. A total of 5482-8692 high-quality reads of 16S

rRNA V3-V5 regions were obtained. Taxonomic analysis

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
using QIIME and RDP classifier found that relative abundance of microbial communities. Microbial

Proteobacteria, Bacteroidetes, Chloroflexi and Firmicutes community in A+OSA system was provided with higher

were the most abundant phyla. Multivariate linear evenness and stability. Moreover, the A+OSA process had

regression analysis indicated that AOA and anammox a significant effect on microbial community structure.

bacteria may be heterotrophic or mixotrophic. Ammonia- Xiao et al. (2015) investigated the microbial

Oxidizing Archaea (AOA) and Ammonia-Oxidizing communities of denitrifying biocathodes yielding current

Bacteria (AOB) showed partially beneficial cooperation of 1 mA (reactor C1) and 1.5 mA (reactor C2) by 454

with anammox genes. Finally, redundancy analysis pyrosequencing. High-quality sequences of 18509 in C1

confirmed the coexistence of anammox, denitrification and 14857 in C2 were generated. The nitrate removal

and DNRA, and revealed that operational parameters efficiencies in C1 and C2 were about 93 and 85%,

highly effect microbial communities. respectively. Classes of Alphaproteobacteria,

Geisen et al. (2015) created a mock community Anaerolineae, and Phycisphaerae may benefit from the

of common free-living soil protists, extracted DNA and performance of current production and nitrate removal.

amplified it in the presence of metazoan DNA using 454 Twenty-nine dominant OTUs accounted for 64 and 65%

high throughput sequencing. This study aimed at of sequences in C1 and C2, respectively.

evaluating whether HTS quantitatively reveals true Benbow et al. (2015) characterized epinecrotic

relative abundances of soil protists and at investigating bacterial communities on vertebrate during decomposition

whether the expected protist community structure is through 454 pyrosequencing and analyzed at phyletic and

altered by the amplification of metazoan-associated protist generic taxonomic resolutions. It was found that the

taxa. All the results showed that profound differences in genera richness significantly increased over

amplification success between protist taxa and an decomposition. Additionally, significant differences in

inevitable co-extraction of protist taxa parasitizing soil bacterial communities between seasons, and among

metazoa obscure the true diversity of free-living soil sampling days within each season were shown, suggesting

protist communities. a succession of these communities.

Zhou et al. (2015) investigated the oxic- Lu, Zhang et al. (2015) investigated bacterial

settling-anaerobic (OSA) sludge reduction process, pathogens and their potential virulence in a sewage

anoxic-OSA (A+OSA) sludge reduction system, and a treatment plant (STP) applying both conventional and

conventional anoxic-oxic (AO) system by 454- advanced treatment processes through 454

pyrosequencing. Results revealed that A+OSA system pyrosequencing, Illumina high-throughput sequencing and

achieved good performances of pollutants removal and metagenomic analysis. Pyrosequencing and Illumina

sludge reduction, as the A+OSA system showed higher sequencing consistently demonstrated that Arcobacter

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
genus was the abundant pathogen. At species level, characterization of the communities. In the anodic

Arcobacter butzleri, Aeromonas hydrophila and Klebsiella biofilm, microorganisms belonging to Geovibrio genus

pneumonia dominated in raw sewage, which was also and purple non-sulfur (PNS) bacteria were found to be

confirmed by quantitative real time PCR. Through most of dominant. While in the cathodic bioflim, the alkaliphilic

the potential pathogens and virulence factors were genus Nitrincola and anaerobic microorganisms belong to

eliminated in the STP, the residual A. Butzleri was still Porphyromonadaceae family were the most abundant

present in the final effluent, which need to be further bacteria.

removed. Goltsman et al. (2015) used community

Ma et al. (2015) used Illumina high-throughput transcriptomics to analyze the microbial diversity of

sequencing to reveal the community structures of nine natural acid mine drainage biofilms. Results showed that

coking wastewater treatment plants for the first time. The 91.8% of the ~254 million Illumina reads mapped to

sludge systems showed a similar community composition rRNA genes were represented in the SILVA database. Up

at each taxonomic level. For the low percentage of the to 159 different taxa, including Bacteria, Archaea and

autotrophic ammonia-oxidizing bacteria and nitrite- Eukaryotes, were identified. Leptospirillum bacteria

oxidizing bacteria, both real-time PCR and fluorescence in generally dominated biofilms, but a wide variety of other

situ hybridization analysies were applied to verify. Nitrospira organisms were also detected at very low

Illumina MiSeq sequencing. Fouhy et al. abundance. The results also indicated that a much larger

(2015) conducted the first Illumina MiSeq sequencing variety of organisms were still not selected from the

investigation into the effects of fecal storage on the biofilm.

microbiota, compared to fresh samples. Culture-based Polka et al. (2015) investigated the bacterial

analysis was also completed. Results showed that no diversity in batches of Salame Piacentino PDO (Protected

significant differences occurred at phylum or family level Designation of Origin), a dry fermented sausage that is

between the treatment groups, as well as in the culture- typical in Italy. Salami samples from 6 different local

based analysis. Diversity analysis indicated that samples factories were analyzed. Culture-based microbiological

clustered based on the individual donor, rather than by analyses and PCR-DGGE were carried out aiming to be

storage group. compared with high-throughput sequencing technologies

Daghio et al. (2015) characterized the microbial (HTS) results. A total of 722,196 high quality sequences

communities operating in a single chamber microbial fuel were obtained and quality screening of raw reads obtained

cell (SCMFC) fed with acetate and inoculated with a by Illumina Miseq sequencing. Thirty-two different

biogas digestate. Illumina sequencing of a fragment of the Staphylococcus and 33 Lactobacillus species were

16S rRNA gene was applied for taxonomic identified in the salami from different producers.

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
 Zhang, Qu et al. (2015) constructed three force of biofilm during its development was characterized,

activated sludge systems stimulated by different aromatics and was found to positively and significantly correlate

for indigo production from indole. Illumina MiSeq with the contents of extracellular polymeric substances

sequencing of 16S rRNA gene amplicons was applied to (EPS). Also, 16S rDNA-based Miseq sequencing was

analyze the microbial community structure and applied to reveal microbial community structure.

composition. Results showed that the bacteria assigned to Sphaerotilus, Zoogloea and Haliscomenobacter were

phylum Proteobacteria, family Comamonadaceae, and predominant in the initial stage.

genera Diaphorobacter, Comamonas and Aquamicrobium Schirmer et al. (2015) conducted a large study

were commonly shared dominant population. Moreover, on the error patterns for the MiSeq based on 16S rRNA

pearson correlation were calculated to discern the amplicon sequencing data. They tested state-of-the-art

relationship between microbial communities and indigo library preparation methods for amplicon sequencing and

yields. showed that the library preparation method and the choice

Liao et al. (2015) investigation the impacts of of primers are the most significant sources of bias and

backwashing on the nutrition removal of the pilot-scale cause distinct error patterns. Furthermore we tested the

biological activated carbon (BAC) filtration system, and efficiency of various error correction strategies and

the biomass and microbial community structure of BAC identified quality trimming (Sickle) combined with error

biofilm. Results showed that backwashing was found to correction (BayesHammer) followed by read overlapping

transitionally improve the removal of DOC, DON and N- (PANDAseq) as the most successful approach, reducing

nitrosamine precursors. MiSeq sequencing analysis substitution error rates on average by 93%.

revealed that backwashing had a strong impact on the

bacterial diversity and community structure of BAC The microbial metagenomics analysis

biofilm, but they could gradually recover with the Recently, researchers started to have concerns

operating time after backwashing. Phylum Proteobacteria about the relationship between community composition

was the largest bacterial group in BAC biofilm. and their functions, based on microbial community

Microorganisms from genera Bradyrhizobium, analysis.

Hyphomicrobium, Microcystis and Sphingobium might Llorens-Mares et al. (2015) explored the links

contribute to the effective removal of nitrogenous organic between composition and functionality in microbial

compounds by drinking water biofilter. communities in the Lake Banyoles karstic area through

Zhu et al. (2015) investigated biofilm properties metagenomics and in silico reconstruction of carbon,

evolution coupled with different ages during the start-up nitrogen and sulfur metabolic pathways that were tightly

period in a moving bed biofilm reactor system. Adhesion coupled through a few bacterial groups. Chlorobiaceae

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
was found to play a major role for nitrogen and carbon several polyester substrates including polylactic acid

fixations. Campylobacterales accounted for a large suggesting that they can be used for polyester

percentage of denitrification genes. In addition, depolymerization.

Callionellales was supposed to be involved in Li, Fei et al. (2015b) constructed a metagenomic

denitrification, iron oxidation and carbon fixation, as well fosmi library containing 8× 104 clones from a soil sample

as biogeochemistry of the iron cycle. Bacteroidates were taken from the stacking place of shrimp and crab shell.

also abundant and had potential for dissimilatory nitrate Twelve clones with chitosanase activity were screened.

reduction to ammonium. The results still showed more Through subsequent construction of a subclone library, 3

detailed perspective on the microbial ecology of anoxic chitosanase positive clones were selected. The gene csnA

environments. was expressed in Escherichia coli Rosettagami, which

Taketani et al. (2015) used the Ion Torrent performed stable activity at a wide pH range from 4.5 to

platform to sequence the metagenome of the rhizosphere 6.5 and was thermostable at 50℃ and 55℃. The enzyme

of a native leguminous plant, in order to understand the was significantly inhibited by various heavy metal ions

dynamic of rhizophere microbial communities. The and SDS, while enhanced by Mn2+, Sr2+, Tweens and

annotation indicated that Actinobacteria and Tritons. In addition, the corresponding recombinant

Proteobacteria were the most abundant groups, and chitosanase CsnA was overexpressed. The recombinant

carbohydrate and protein metabolisms were the dominant chitosanase could hydrolyze chitosan efficiently into chi-

functional groups. In the rainy season, the communities tosan dimer, trimer, tetramer, pentamer and hexamer.

carried genes related to carbohydrate and amino acid Roume et al. (2015) carried out a comparative

metabolisms, probably due to the abundance of organic integrated omic analysis to elucidate functional

matter. differences between seasonally distinct oleaginous mixed

Tchigvintsev et al. (2015) screened three marine microbial communities (OMMCs) sampled from an

metagenome gene libraries for esterase activity, so as to anoxic BWWT tank. The analysis included

explore the natural enzyme diversity of cold environments metagenomics, metatranscriptomics and metaproteomics.

and identify new carboxylesterases. Five esterases were A computational framework for the reconstruction of

selected from biochemical characterization. All five community-wide metabolic networks from multi-omic

esterases retained high activity at 5℃ indicating that they data was also developed. Results showed that genes

are cold-adapted enzymes. The activity of MGS0010 exhibiting high expression relative to gene copy

increased more than two times in the presence of high abundance included genes that involved in glycerolipid

concentrations of NaCl or KCl, while the other four were metabolism, particularly triacylglycerol lipase, encoded

inhibited. The metagenomic esterases also hydrolyzed by known lipid accumulating populations, e.g.,

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
Candidatus Microthrix parvicella. Genes with a high enabled efficient identification of an Ubiquinol-

relative gene expression and topologically important cytochrome reductase binding protein (UQCRB)

positions in the network included genes that involved in homologue, a novel major allergen designated Der f 24,

nitrogen metabolism and fatty acid biosynthesis, encoded through a multi-omic approach.

by Nitrosomonas spp. and Rhodococcus spp. Dupont et al. (2015) examined transitions in

community genomic features and biogeochemical

Microbial transcriptome analysis processes in surface and substrate chlorophyll maximum

Based on the analysis of the functions of (SCM) microbial communities across a trophic gradient

microbial community, the analysis of gene expression from mesotrophic waters to the oligotrophic Pacific.

from the view of RNA can clearly indicate the adaptive Results showed that pelagophytes, very similar to

mechanism of microbial community and the various Pelagomonas sp. CCMP1756, appeared to be a major

changes of functional genes when it comes to constituent of SCM communities and may dominate

environmental change. nitrate uptake and reduction.

Klein et al. (2015) identified a minisatellite Le Bihan et al. (2015) examined the adaptive

contained within a miniature inverted-repeat transposable response of enterohemorrhagic Escherichia coli (EHEC)

element (MITE) in Porphyromonas gingivalis. The P. to the intestinal milieu conditioned by a human

gingivalis minisatellite and associated MITE was named microbiota. It was based on a transcriptomic analysis on

BrickBuilt. Segments of BrickBuilt contained promoter the EHEC strain EDL933 incubated in vitro in the sterile-

elements with bidirectional transcription capabilities. filtrated cecal content of human microbiotaassociated rats

They also performed a bioinformatic analysis of (HMC) compared with EDL933 incubated in the sterile-

BrickBuilt by means of existing whole genome filtrated cecal content of germ-free rat (GFC). The results

sequencing, microarray and RNAseq data, as well as showed that the microbiota was able to repress EHEC

performing in vitro promoter probe assay. Results virulence gene expression, such as those involved in the

indicated that these elements may play an important role utilization of sugars, glycerol, N-acetylneuraminic acid,

in facilitating genome evolution as well as modulating the amino acids and secondary metabolites.

transcriptional regulatory system. Rurangwa et al. (2015) assessed the impact of

Chan, Ji et al. (2015) produced a D farinae two dietary inclusion levels of a novel protein meal

genome draft, which revealed the full gene structures of (NPM) of animal origin on the gastrointestinal tract (GIT).

20 canonical mite allergens and 7 noncanonical allergen Microbial development was assessed through 16S rRNA

homologues. Enterobacter species is the predominant gene-based microbial composition profiling by

genus in the D farinae gut microbiome. The genome draft pyrosequencing. Differentially expressed genes in the GIT

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
were demonstrated by whole transcriptome sequencing expression patterns, for example, the colon-enriched

(mRNAseq). Results showed that the high iron content of expression of NXPE family member 1, and NLR family,

NPM may lead to an increased relative abundance of pyrin domain-containing 6 (NLRP6) expression.

bacteria with increased iron metabolism. The GIT of Nobu et al. (2015) employed state-of-the-art

larvae fed at higher NPM level was also more active in omic tools to generate draft genomes and transcriptomes

transmembrane ion transport and protein synthesis. An for uncultivated organisms spanning 15 phyla in a

mRNAseq analysis did not reveal a major activation of methanogenic environment. Results showed that the

genes involved in the immune response or indicate uncultivated organisms might interact to form an intricate

differences in iron uptake and homeostasis with high syntrophy-supported food web with Pelotomaculum and

inclusion level of NPM. methanogens to metabolize catabolic by-products and

Dedeine et al. (2015) focused on transcriptomes detritus, whereby facilitating holistic terephthalate (TA)

of secondary reproductive females. Thirteen mineralization to CO2 and CH4.

transcriptomes were used. After transcriptome assembly Wei et al. (2015) performed an Illumina-based

and read mapping, they examined the expression of deep sequencing of mRNA to understand the gene

interspecific variation of genes, and detected 18,323 expression profile in the mal accessory glands and

orthologous gene clusters. Functional annotation and ejaculatory duct (MAG/ED) of the oriental fruit fly

taxonomic assignment were performed on a total of Bactrocera dorsalis. Results showed that 20,419 unifenes

41,287 predicted contigs in the three termite species. were functionally annotated to known proteins in a

Results showed that functional categories of genes were database. Many genes were involved in immunity, the

comparable, and 9 cellulases and a total of 79 contigs inducible expression of which was confirmed by qRT-

potentially involved in 11 enzymatic activities used in PCR analysis when insects were challenged with

wood metabolism. immunity-inducible factors. In addition, some important

Gremel et al. (2015) applied a genome-wide reproductive genes were identified.

analysis based on transcriptomics and antibody-based Chieng et al. (2015) examined the expression

protein profiling to identify genes that are expressed in a profile of B. pseudomallei T6SS six gene clusters during

specific manner within the human gastrointestinal tract infection of U937 macrophage cells. Three highly

(GIT). Results revealed that 51 genes showed enriched expressed T6SS genes, tssj-4, hcp1 and tssE-5, were

expression in either one of the GIT tissues and an expressed as recombinant proteins and screened by

additional 83 genes were enriched in two or more GIT western analysis and enzyme-linked immuno sorbent

tissues. In this study, they characterized various well- assay (ELISA). Only Hcp1 was reactive and further

known and poorly studied genes with validated protein evaluated. The detection of aniti-Hp1 antibodies in

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patients’ sera indicating the presence of B. pseudomallei the flight strains. The expression level of more than 1000

highlights the potential of Hcp1 to be further developed as genes altered in flight strains. Genes involved in

a serodiagnostic marker for melioidosis. chemotaxis, lipid metabolism and cell motility express

Kopf et al. (2015) compared the primary differently. Moreover, the two flight strains also differed

transcriptomes of the cyanobacteria Synechocystis sp. Pcc extensively from each other in terms of metabolism,

6714 and PCC6803 under 10 different conditions. Results transcriptome and proteome, indicating the impact of

showed that these strains were closely related. Non-coding space environment on individual cells is heterogeneous

transcripts were identified as the most dynamic and probably genotype-dependent.

component of the transcriptome. They also identified a

class of mRNAs that originated from a sRNA that Microbial protein group

accumulated as a discrete and abundant transcript while Microbial proteomics directly indicate the

also serving as the 5’UTR. Such a sRNA/mRNA structure variety of proteins expressed and the corresponding

represented another way for bacteria to remodel their functions, from the perspective of protein, when microbial

transcriptional network. communities adapt to environmental change.

Hovde et al. (2015) reported the draft genome Jia et al. (2015) studied the global proteome

sequence of Chrysochromulina tobin (Prymnesiales), and responses of V. Metschnikovii under cold stress by nano-

transcriptome data. The nuclear genome of C. tobin is flow ultra-high-performance liquid chromatography

small, compact and encodes approximately 16,777 genes. coupled to a quadrupole Orbitrap mass spectrometer. A

The C. tobin genome harbors the first hybrid polyketide total 2066 proteins were identified, the functional

synthase/non-ribosomal peptide synthase gene complex categorization of which showed distinct differences

reported from an algal species, and encodes potential between cold-stressed and control cells. Quantitative

antimicrobial peptides and proteins involved in multidrug reverse transcription polymerase chain reaction analysis

and toxic compound extrusion. was also performed to determine the mRNA expression

Li, Chang et al. (2015) conducted detailed levels of seventeen cold stress-related genes.

genomics, transcriptomic and proteomic studies on E.coli Zhang, Guo et al. (2015) evaluated the diversity

that experienced 17 days of spaceflight, to better of morphologies, microbial communities, extracellular

understand the mechanism how E.coli overcomes harsh proteins and polysaccharides between the biofilm and

environments and the related factors. Results showed that suspended sludge (S-sludge), in order to investigate the

the flight strains differed from the control in the utilization mechanisms of governing the differences between them in

of more than 30 carbon sources. Two single nucleotide terms of physicochemical properties and process

polymorphsms (SNPs) and one deletion were identified in performances. Results showed that the denitrification was

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
much more extensive performed in S-sludge than biofilm, investigate the functional role of closely related species

while many microbial cells in the biofilm maintained their and strains and to determine if organisms are capable of

structure stability. An extracellular protein that could both aerobic and anaerobic growth. Proteome extracts

promote the cell adhesion was observed only in the from fecal samples were processed and peptides were

biofilm, but more extracellular proteins related to catalytic identified based on information predicted from the

activity to decrease the compactness of floc was found in genome sequences for the dominant organisms, Serratia

the S-sludge. Moreover, the monosaccharide compositions and the two Citrobacter strains. These organisms are

from the two aggregates were various. facultative anaerobes, and proteomic information

Xiong, Abraham et al. (2015) studied the field indicated the utilization of both aerobic and anaerobic

of human gut microbiome metaproteomics through metabolisms. Results also revealed the differences in

characterizing systems ranging from low-complexity motility and chemotaxis functions of coexisting

model gut microbiota in gnotobiotic mice, to the emerging Citrobacter strains. Moreover, Citrobacter strains showed

gut microbiome in GI tract of newborn human infants, and a community-essential role in vitamin metabolism and a

finally to an established gut microbiota in human adults. predominant role in propionate production.

Microbial and human proteins were extracted and Bastida et al. (2015) evaluated the impacts of

measured by high performance MS-based proteomics petroleum contamination and the bioremediation potential

technology, with the aim to characterize the human gut of compost amendment, to enhance the microbial activity

microbiota functions and metabolic activities in semiarid soils. In this study, phospholipid fatty-acids

Kohrs et al. (2015) applied centrifugal and metaproteomics were analyzed. Petroleum pollution

fractionation for fractionation of two biogas sludge induced an increase of proteobacterial proteins during the

samples to analyze proteins extracted from crude fibers, contamination, while the relative abundance of

suspended microorganisms, and secreted proteins in the Rhizobiales lowered. The compost assisted bioremediation

supernatant using a gel-based approach followed by LC- promoted the removal of polycyclic aromatic

MS/MS identification. This method was beneficial to the hydrocarbons (PAHs) ad alkanes, mainly driven by

quality of SDS-PAGE and the identification of peptides Sphingomonadales and uncultured bacteria that showed an

and proteins, however, it influenced significantly the increased abundance of catabolic enzymes. In addition,

number and the types of proteins identified in the only 0.55% of the metaproteome of the compost-treated

microbial metaproteomes. soil was involved in biodegradation processes, which

Brooks et al. (2015) implemented a high- achieved great performance.

throughput mass spectrometry-based proteomics approach Chen et al. (2015) conducted comparative

to identify proteins in fecal samples, in order to metagenomics and metatranscriptomics on microbial

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
assemblages collected from four geochemically distinct Statistical analyses provided useful information on

acid mine drainage sites (AMD). Taxonomic analysis biological reproducibility and replicate necessity. No

uncovered high microbial biodiversity of the extremely newly expressed proteins were found and 62 proteins were

acidophilic communities, and the abundant taxa of detected following coherent and plausible expected trends

Acidithiobacillus, Leptospirillum and Acidiphilium with in terms of presence and intensity change.

high transcriptional activities. Community-wide

comparative analyses showed that AMD microorganisms Microbial metabolomics

expressed genes involved in multiple in situ functional Microbial metabolomics is an important

activities to adapt different environment conditions, research direction of molecular biology in the future.

including low-pH adaptation, carbon, nitrogen and Microbial metabolomics expands the multi-scale research

phosphate assimilation, energy generation, environmental on electron transfer pathways of microorganisms,

stress resistance, and other functions. metabolic function, metabolic network and so on, from the

Zhang, Shen et al. (2015) analyzed the origins original research on metabolites of microorganisms. The

and functions of proteins in extracellular polymeric research of microbial metabolomics are based on DNA,

substances extracted from anaerobic, anoxic, and aerobic RNA, protein, and secondary metabolites.

sludge of a wastewater treatment plant. Shotgun Xu et al. (2015) investigated the biochemical

proteomics were applied to identify extracellular proteins, compositional changes occurring during the microbial

and 130,138 and 114 proteins in anaerobic, anoxic and fermentation process (MFP) of Fu brick tea (FBT) based

aerobic samples were classified, respectively. The results on non-targeted LC-MS. FBT is fermented with fungi

revealed that most proteins were from cell and cell parts, during the manufacturing process. It was confirmed that

and extracellular proteins in activated sludge play main MFP significantly influenced the quality of FBT. The

roles in mutivalence cations and organic molecules non-targeted LC-MS analysis clearly distinguished FBT

binding, as well as in catalysis and degradation. The with other tea samples and highlighted some metabolic

catalytic activity proteins were more widespread in pathways during MFP including caffeine produced by

anaerobic sludges, and this resulted in structure molds, B ring fission catechins, characteristic elevation of

differences between anaerobic and aerobic sludges. gallic acid and so on.

Buttiglieri et al. (2015) studied pharmaceutical Warren (2015) examined methods for extracting

trace compound degradation pathways in wastewater. total organic N monomers by contrasting chloroform gas

Proteomics reliability dealing with activated sludge spiked fumigation with chloroform direct extraction, and

with a pharmaceutical compound (ibuprofen) was examining whether it is possible to extract soil with two

investigated over time and at different concentrations. methods that combine quenching of metabolic activity

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
with extraction, namely cold methanol/chloroform/water strains of the non-Aspergillus species were observed. One

and hot aqueous ethanol. Absolute concentration of 32 out of the eight compounds, Leu-Glu-Leu-Glu, was found to

of the 42 quantified organic N monomers were be a novel and first linear tetrapeptide observed in

significantly different between soil extracted by Aspergillus species. Two other compounds, hydroxy-

chloroform gas fumigation and chloroform direct (sulfooxy)benzoic acid and (sulfooxy)benzoic acid might

extraction, which were probably due to oxidation, possess anti-inflammatory properties.

hydrolysis and deamidation during chloroform gas Quinn et al. (2015) used molecular networking-

fumigation. However, the extraction with based metabolomics to investigate the chemistry of cystic

methanol/chloroform/water is ineffective at extracting fibrosis (CF) sputa and assess how the microbial

strongly cationic compounds, while extraction with hot molecules detected reflect the microbiome and clinical

aqueous ethanol was unsuccessful with soil. culture history of the patients. This study demonstrated

Lu, Liu et al. (2015) investigated the metabolic that the chemical makeup of CF sputum was a complex

differences and regulation mechanisms between a high milieu of microbial, host and xenobiotic molecules.

glucoamylase-producing strain A. Niger DS03043 and its Detection of a bacterium by clinical culturing and 16S

wild-type parent strain A.niger CBS513.88 via an rRNA gene profiling did not necessarily reflect the active
13C
integrated isotope-assisted metabolomics and production of metabolites from that bacterium in a sputum

metabolic flux analysis approach. Results indicated that an sample.

imbalance of NADH formation and consumption led to Guard et al. (2015) evaluated the fecal

the accumulation and secretion of organic acids in A. microbiome, fecal concentrations of short-chain fatty

niger CBS513.88. The significantly reduced by-product acids (SCFAs), as well as serum and urine metabolites in

formation rates resulted in an increased energy utilization healthy dogs and dogs with acute diarrhea. The fecal

efficiency and specific growth rate in A. niger DS03043. microbiome, SCFAs, and serum/urine metabolite profiles

To enable a more efficient enzyme production and faster were characterized by 454-pyrosequencing of the 16S

cell growth, the total amino acids pool size, the ratio of rRNA genes, GC/MS, and untargeted and targeted

ATP to AMP and the flux channeled into the PP pathway metabolomics approach using UPLC/MS and HPLC/MS,

were obviously improved in A. niger DS03043. respectively. The microbial communities were

Lee et al. (2015) compared the metabolomic significantly different between dogs with acute diarrhea

profiles of the culture supernatants of 30 strains of six and healthy dogs. It was found that the genus Clostridium

pathogenic Aspergillus species and 31 strains of 10 non- was over-represented in dogs with acute diarrhea, and the

Aspergillus fungi, from which, eight compounds present concentration of fecal propionic acid were significantly

in all strains of the six Aspergillus species but not in all decreased, which were correlated to a decrease in

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Water Environment Research, Volume 88, Number 10—Copyright © 2016 Water Environment Federation
Faecalibacterium. In addition, results revealed antibiotics revealed that our analytical method is capable

overrepresentations of genes for tansposase enzymes and of displaying metabolic changes linked to a shift in

methyl accepting chemotaxis proteins, and significantly microbial diversity.

decreased in serum concentration of kunurenic acid and Acknowledgements

urine concentration of 2-methyl-1H-indole and 5- The authors gratefully acknowledge the support

Methoxy-1H- indole-3- carbaldehyde in acute diarrhea. of the Project supported by the Funds for Creative

Zhang, Xie et al. (2015) presented a novel Research Groups of China (Grant No. 51121062),

nanoparticle-based strategy that differentiates between the Supported by State Key Laboratory of Urban Water

metabolites based on their electric charge, in order to Resource and Environment (Harbin Institute of

enhance the comprehensive and accurate identification of Technology) (No. 2015TS07).

the underlying metabolites. By adding electrically charged

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Etyemez, M., Balcázar, J. L. (2015) Bacterial Community Hovde, B. T., Deodato, C. R., Hunsperger, H. M., Ryken,

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Microbial Metagenomics. ISME J., 9(7), 1648-1661. Identification of Initial and Spoilage Microbiota of

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