A novel cyanobacterial geosmin producer, revising GeoA distribution and dispersion patterns in Bacteria

Authors

Catarina Churro, Ana P. Semedo-Aguiar, Alexandra D. Silva, Jose B. Pereira-Leal & Ricardo B. Leite

Cyanobacteria are ubiquitous organisms with a relevant contribution to primary production in all range of habitats. Cyanobacteria are well known for their part in worldwide occurrence of aquatic blooms while producing a myriad of natural compounds, some with toxic potential, but others of high economical impact, as geosmin. We performed an environmental survey of cyanobacterial soil colonies to identify interesting metabolic pathways and adaptation strategies used by these microorganisms and isolated, sequenced and assembled the genome of a cyanobacterium that displayed a distinctive earthy/musty smell, typical of geosmin, confirmed by GC-MS analysis of the culture’s volatile extract. Morphological studies pointed to a new Oscillatoriales soil ecotype confirmed by phylogenetic analysis, which we named Microcoleus asticus sp. nov. Our studies of geosmin gene presence in Bacteria, revealed a scattered distribution among Cyanobacteria, Actinobacteria, Delta and Gammaproteobacteria, covering different niches. Careful analysis of the bacterial geosmin gene and gene tree suggests an ancient bacterial origin of the gene, that was probably successively lost in different time frames. The high sequence similarities in the cyanobacterial geosmin gene amidst freshwater and soil strains, reinforce the idea of an evolutionary history of geosmin, that is intimately connected to niche adaptation.

First report of geosmin and 2-methylisoborneol (2-MIB) in Dolichospermum and Oscillatoria from Vietnam

Authors

Thanh-Luu Pham, Manh Ha Bui, Mark Driscoll, Kazuya Shimizu & Utsumi Motoo

Cyanobacteria are the major producers of the taste and odour compounds geosmin and 2-methylisoborneol (2-MIB) in drinking water and fish worldwide. In recent years, the outbreaks of cyanobacteria blooms in Tri An and Dau Tieng Reservoirs, Vietnam have led to an increase attention in taste and odour problems. In the present study, the two off-flavour compounds geosmin and 2-MIB were found in field samples and 50 isolated strains of planktonic cyanobacteria from Dau Tieng and Tri An Reservoirs. They were screened for the first time using headspace solid-phase microextraction and gas chromatography–mass spectrometry (HPME GC–MS). The ability to produce off-flavour compounds was determined by the presence of two molecular markers: geoA and mib. Results showed that mean geosmin and 2-MIB concentrations ranged from 13.3 to 9.6 ng/L and 4.9 to 101.7 ng/L in field samples, respectively. A total of five geosmin-producing strains [with the highest concentration: 272.9 ng/g wet weight (ww)] and nine 2-MIB-producing strains (with the highest concentration: 362.1 ng/g ww) belonging to two cyanobacteria genera Dolichospermum and Oscillatoria were identified. Four of the geosmin producers also produced 2-MIB. The 16S rRNA maximum likelihood (ML) tree showed a high diversity of the isolated strains that represented different morphotypes and genotypes. The BLAST searches revealed that the 16S rRNA gene sequence obtained in this study shared the similarity values ranging between 96.46 and 99.37% compared to the available sequences from GenBank. This is the first report of off-flavour compound producing cyanobacteria in Vietnamese waters. The combination of HPME GC–MS and polymerase chain reaction (PCR) provides a robust tool for reliably characterizing the off-flavour compound. Our results highlight that taste and odour-producing cyanobacteria belonging to Chroococcales in Vietnamese waters are unexplored and require further investigation.

 

Effects of pre, post, and simultaneous loading of natural organic matter on 2-methylisoborneol adsorption on superfine powdered activated carbon: Reversibility and external pore-blocking

Authors

Akiko Nakayama, Asuka Sakamoto, Taku Matsushita, Yoshihiko Matsui, Nobutaka Shirasaki

Three different natural organic matter (NOM)-loading methods were compared for the adsorptive removal of 2-methylisoborneol (MIB) by superfine powdered activated carbon (SPAC) and conventionally-sized powdered activated carbon (PAC). The three NOM-loading methods were: NOM adsorption followed by MIB (MIB adsorption on NOM-preloaded carbon), MIB adsorption followed by NOM (MIB adsorption on NOM post-loaded carbon), and simultaneous NOM and MIB loading (MIB adsorption on NOM-simultaneously loaded carbon). MIB removals were similar for the smaller-sized carbon (SPAC) at higher AC dosages and at lower initial NOM concentrations. The similar MIB removals indicate direct site competition between MIB and NOM with MIB adsorption reversibility (complete desorption of MIB by NOM). At lower AC doses, especially for PACs, and at higher initial NOM concentrations, the adsorption of MIBs depended on the sequence of MIB or NOM adsorption. MIB removal was lowest for the NOM-preloaded carbon, followed by NOM-simultaneously loaded carbon. The highest MIB removal was achieved by post-loading of NOM, indicating that the adsorption is irreversible. MIB adsorption on SPAC was more reversible than on PAC, although the pore size distributions of the two carbons were similar. The high degree of adsorption irreversibility for PAC compared with SPAC indicated that pore blocking occurs due to NOM loading at the PAC particle surface. Images of the external adsorption were obtained using isotope mapping and ¹⁵N-labeled effluent organic matter.

Pyrazines: A diverse class of earthy-musty odorants impacting drinking water quality and consumer satisfaction

Authors

Chunmiao Wang, Jianwei Yu, Daniel L. Gallagher, Julia Byrd, Wenchuo Yao, Qi Wang, Qingyuan Guo, Andrea M. Dietrich, Min Yang

The presence of earthy-musty odors in drinking water is a major concern for water suppliers and consumers worldwide. While geosmin and 2-methylisoborneol are the most studied earthy-musty odor-causing compounds, pyrazine and its alkyl and methoxy compounds possess similar odors and are widely distributed in nature, foods, and beverages. In this study, odor characteristics of pyrazines and their presence in natural and treated waters were determined. Pyrazine, 2,6-dimethyl-pyrazine (DMP), 2,3,5-trimethyl-pyrazine (TrMP), 2-ethyl-5(6)-methyl-pyrazine (EMP), 2,3,5,6-tetramethyl-pyrazine (TeMP), 2-isobutyl-3-methoxy-pyrazine (IBMP) and 2-isopropyl-3-methoxy-pyrazine (IPMP) were measured in source and finished drinking water across China. 2-Methoxy-3,5-dimethyl-pyrazine (MDMP), IBMP, and IPMP were investigated in rivers in Virginia, USA. The results showed that “musty” and “sweet” were the most common descriptors for pyrazine, DMP, MDMP, TrMP, and TeMP. While IBMP and IPMP were never detected in 140 source or drinking water samples from across China, pyrazine, DMP, MDMP, TrMP, and TeMP occurred throughout with concentrations of n.d.−62.2 ng/L-aq in source water and n.d.−39.6 ng/L-aq in finished water. IBMP, IPMP, and MDMP were present in two Virginia rivers; MDMP occurred in 18% of the samples with concentrations of n.d.−4.4 ng/L, many of which were above the aqueous odor threshold of 0.043 ng/L MDMP. The removal efficiencies through conventional water treatment were poor, ranging from negative removals to ∼10%. Advanced oxidation water treatment could only remove EMP and TrMP. The widespread presence of earthy-musty-sweet pyrazines in source and drinking waters on two continents, their poor removal during water treatment, and ng/L odor threshold concentrations confirm their potential to be T&O issues for consumers.

Co-occurrence of microcystin- and geosmin- producing cyanobacteria in the Tri An Reservoir, a drinking-water supply in Vietnam

 

Authors

Pham, Thanh-Luu; Tran, Thi Hoang Yen; Hoang, Nghia Son; Ngo, Xuan Quang; Tran, Thai Thanh

The present study aimed to investigate the occurrence and distribution of microcystin-(MC) and geosmin-producing cyanobacteria in the Tri An Reservoir, a drinking-water supply in Southern Vietnam. The MC concentration was measured using high-performance liquid chromatography (HPLC). Geosmin production was screened using headspace solid-phase microextraction (HPME) and gas chromatography/mass spectrometry (GC/ MS). The ability of cyanobacteria to produce MC and geosmin was determined by the presence of two molecular markers: mcyA and geoA, respectively. A maximum MC concentration of 207.6 ± 14.2 μg g ^–1 dry weight (DW) was detected from an isolated Microcystis aeruginosa (Kützing) (strains TAMA1), and the maximum geosmin concentration of 114.2 ± 6.1 ng g ^–1 wet weight (WW) was detected from an isolated Dolichospermum circinale (Rabenhorst) (strains TADC4); MC and geosmin were also observed in water-bloom samples, with the highest concentrations being 463.6 ± 34.7 μg g ^–1 DW and 148.5 ± 9.8 ng g ^–1 WW, respectively. These results represent the first molecular evidence of the co-occurrence of MC- and geosmin-producing cyanobacteria in a drinking-water-supply reservoir in Vietnam. Given the success of the molecular techniques and chemical analysis used, we propose that multiplex polymerase chain reaction assays, HPLC, and HPME GC/MS can be reliable tools for investigating MC and geosmin in water-bloom and cyanobacterial samples. Further, we propose that they can be used to create early-warning systems for harmful secondary metabolites produced by cyanobacteria in drinking-water supplies.

 

Biological pretreatment: An innovative approach to addressing taste and odor

Authors

Jess Brown Jennifer Nyfennegger Yong Ang Mark Simpson Bruce MacLeod Olga Wolanin Katherine Gilmore

The presence of objectionable taste‐and‐odor (T&O) compounds in surface water supplies is a common problem facing drinking water utilities across the country and worldwide. While there are several viable T&O treatment options, including adsorption, biotransformation, and advanced oxidation, no single option fits all applications or is without potential limitations. Through bench‐ and pilot‐scale testing, this work developed and evaluated high‐rate biological roughing filtration as a promising alternative for geosmin and 2‐methylisoborneol abatement to help utilities minimize T&O complaints without straining their annual operating budgets. Testing showed that biological roughing filtration can effectively treat a wide range of raw water T&O levels using short contact times, and the intermittent presence of T&O compounds did not appear to appreciably affect removal efficacy. Pilot‐testing results were used to develop the design criteria for a full‐scale 54‐mgd biological roughing filter that is currently treating surface water in Manatee County, Florida.

Molecular Probes to Evaluate the Synthesis and Production Potential of an Odorous Compound (2-methylisoborneol) in Cyanobacteria

Authors

Keonhee Kim, Youngdae Yoon, Hyukjin Cho and Soon-Jin Hwang

The volatile metabolite, 2-Methylisoborneol (2-MIB) produced by cyanobacterial species, causes odor and taste problems in freshwater systems. However, simple identification of cyanobacteria that produce such off-flavors may be insufficient to establish the causal agent of off-flavor-related problems as the production-related genes are often strain-specific. Here, we designed a set of primers for detecting and quantifying 2-MIB-synthesizing cyanobacteria based on mibC gene sequences (encoding 2-MIB synthesis-catalyzing monoterpene cyclase) from various Oscillatoriales and Synechococcales cyanobacterial strains deposited in GenBank. Cyanobacterial cells and environmental DNA and RNA were collected from both the water column and sediment of a eutrophic stream (the Gong-ji Stream, Chuncheon, South Korea), which has a high 2-MIB concentration. Primer sets mibC196 and mibC300 showed universality to mibC in the Synechococcales and Oscillatoriales strains; the mibC132 primer showed high specificity for Pseudanabaena and Planktothricoides mibC. Our mibC primers showed excellent amplification efficiency (100–102%) and high correlation among related variables (2-MIB concentration with water RNA r = 689, p < 0.01; sediment DNA r = 0.794, p < 0.01; and water DNA r = 0.644, p < 0.05; cyanobacteria cell density with water RNA and DNA r = 0.995, p < 0.01). These primers offer an efficient tool for identifying cyanobacterial strains possessing mibC genes (and thus 2-MIB-producing potential) and for evaluating mibC gene expression as an early warning of massive cyanobacterial occurrence.

Interspecific competition between Microcystis aeruginosa and Pseudanadaena and their production of T&O compounds

Authors

Kejia Zhang, Renjie Pan, Zhang Luo, Tuqiao Zhang, Jiajia Fan

Microcystis aeruginosa and Pseudanabaena are two common cyanobacterial species/genus and they can occur coincidently in many eutrophic lakes globally. These two cyanobacteria could produce Taste & Odor (T&O) compounds, and their production of T&O compounds might be changed when they are present coincidently. The amounts of T&O compounds and their producers may influence the effectiveness of water treatment processes. Therefore, the mutual interactions between Microcystis aeruginosa (FACHB-905, M) and Pseudanabaena sp. (FACHB-1277, P) on T&O compounds in co-cultures were evaluated in this study. Different initial cell concentrations of M and P, with ratios of M:P = 1:1, M:P = 1:2 and M:P = 2:1 were applied in the co-cultures. The growth of M was enhanced under all of the cyanobacterial cell ratios. The growth of P was enhanced under the ratio of M:P = 1:1, while it was inhibited under the ratios of M:P = 1:2 and M: P = 2:1. In addition, the growth of the two cyanobacteria and their production of β-cyclocitral and 2-methylisoborneol (2-MIB) in the filtrate of P were higher than those in the filtrate of M, which may be attributed to their associated secondary metabolites. The cell integrity and photosynthetic capacity of the two studied cyanobacteria are greatly affected by exposure to β-cyclocitral and 2-MIB. The results showed that β-cyclocitral and 2-MIB had the allelopathic effects on the two cyanobacteria species which might influence the composition of co-existing cyanobacteria and their production of T&O compounds.

Causality analysis and prediction of 2-methylisoborneol production in a reservoir using empirical dynamic modeling

Authors

Manna Wang, Chihiro Yoshimura, Ayman Allam, Fuminori Kimura, Takamitsu Honma

2-Methylisobornel (MIB) is one of the most widespread and problematic biogenic compounds causing taste-and-odor problems in freshwater. To investigate the causes of MIB production and develop models to predict the MIB concentration, we have applied empirical dynamic modeling (EDM), a nonlinear approach based on Chaos theory, to the long-term water quality dataset of Kamafusa Reservoir in Japan. The study revealed the dynamic nature of MIB production in the reservoir, and determined causal variables for MIB production, including water temperature, pH, transparency, light intensity, and Green Phormidium. Moreover, EDM established that the system is three-dimensional, and the approach found elevated nonlinearity (from 1.5 to 3) across the whole study period (1996–2015). By taking only one or two candidate predictors with varying time lags, multivariate models for predicting MIB production (best model: r = 0.83, p < 0.001, root mean squared error = 3.1 ng/L) were successfully established. The modeling approach used in this study is a powerful tool for causality identification and odor prediction, thus making important contributions to reservoir management.

Influence of Environmental Factors on the Production of MIB and Geosmin Metabolites by Bacteria in a Eutrophic Reservoir

Authors

Nicolas A. Clercin, Gregory K. Druschel

Occurrences of odorous bacterial metabolites, 2‐methylisoborneol (MIB) and geosmin (GSM), in drinking water supply reservoirs are considered as a nuisance by the water industry and a source of complaints from customers. In Eagle Creek Reservoir, routine monitoring programs of MIB and GSM highlight intense odorous outbreaks during the spring season when high inflow discharges occur. Cyanobacteria have always been assumed to be source of these metabolites even if no known producers are present in raw water. A copper‐based algaecide is often used to terminate the metabolite production and the algal growth in the reservoir. The current study was designed to investigate and identify other biological sources involved in the biosynthesis of MIB and GSM metabolites as well as environmental factors that could be important triggers for the growth of bacterial producers. The community structure of the bacterioplankton was determined using a 16S rRNA gene sequencing technique, which showed that not only Cyanobacteria but Actinobacteria also were involved in the reservoir internal production. Planktothrix species was identified as the main source of GSM (p < 0.001) while Streptomyces (Actinobacteria) was very likely responsible of MIB (p < 0.01). Application of an algaecide disrupted GSM and the growth of Planktothrix but was less effective against MIB and Streptomyces. Statistical analyses revealed that MIB‐ and GSM‐causing bacteria were found abundant when the water was enriched with nitrogen, temperature cooler, and the water column mixed.