**1. Introduction**

The phototrophic purple bacteria are widely distributed in nature and play important roles in global carbon, nitrogen, and sulfur cycles. Sunlight-exposed stagnant water bodies in natural and engineered environments that contain sulfide and/or high-strength organic matter are rich sources of the phototrophic purple bacteria, which often exhibit massive growth, as seen as red, pink, and brown blooms and microbial mats. The massive development of the phototrophic purple bacteria can be seen in specific habitats [1], including meromictic lakes [2–6], lagoons in intertidal zones [7,8], salt marsh and lakes [9,10], shallow soda pans [11], and wastewater stabilization ponds [12–18]. Naturally occurring massive blooms are mainly caused by the proliferation of the purple sulfur bacteria (PSB), which comprise the family *Chromatiaceae* within the class *Gammaproteobacteria*. On the other hand, it is believed that the purple nonsulfur bacteria (PNSB), belonging to *Alpha*- and *Betaproteobacteria*, rarely form colored blooms in the environment; in fact, there has been only scattered information about the involvement of PNSB in blooming phenomena.

As a rare case, Okubo et al. [19] reported that a swine wastewater ditch allowed PNSB to exclusively develop into red microbial mats. This massive development was possibly achieved under specific conditions characterized by the exposure to light and air, the presence of high-strength organic acids, and the absence of sulfide. Also, *Rhodovulum* (*Rdv*.) *strictum* and some other marine PNSB related to *Rhv. sulfidophilum* were isolated from colored blooms in coastal mudflats and tide pools [20,21]. Although these previous reports sugges<sup>t</sup> the potential of PNSB to naturally develop massive populations under particular conditions, it is still uncertain how many PNSB populations co-exist with PSB in natural blooming communities and what the factors are that allow for the massive growth of PNSB in the environment.

This study was undertaken to determine how many populations of PNSB occur in colored blooms and microbial mats developing in coastal and wastewater environments. In order to obtain quantitative and qualitative information on PNSB populations, we used a polyphasic approach by photopigment and quinone profiling, *pufM* gene-targeted quantitative PCR (qPCR), and *pufM* gene clone library analysis, in addition to conventional cultivation-dependent approaches. Quinone profiling is a chemotaxonomic biomarker method useful for roughly determining microbial populations in terms of quantity and quality [22,23]. The clone library analysis of *pufLM* genes, encoding the L and M subunits of photochemical reaction center proteins, is useful to classify PSB and PNSB phylotypes in mixed populations for which 16S rRNA genes are not suitable as phylogenetic markers [8,10,19,24–26]. We also determined the genome sequence of *Rhodovulum* sp. strain MB263, which was previously isolated from a pink-blooming mudflat [21], and the type strain of *Rdv. sulfidophilum*. Based on the results of these analyses, we discuss the environmental conditions under which PNSB can overgrow PSB and co-existing chemoheterotrophic bacteria and the importance of *Rhodovulum* members as the major PNSB populations in coastal environments.

#### **2. Materials and Methods**

#### *2.1. Studied Sites and Samples*

Colored bloom and mat samples were collected from different environments in Japan from 2002 to 2014 (Table 1). The studied sites, geographical coordinates of the locations, and sample designations were as follows: mudflat (Yatsuhigata, Narashino (35◦40-32" N, 140◦00-17" E); samples Y1–Y3), tide pools (Jogashima, Yokosuka (35◦07-52" N, 139◦37-06" E); samples J1–J3), and wastewater ditches in Kosai (34◦41-23" N, 137◦29-27" E; sample D1), Shizuoka (35◦01-00" N, 138◦26-57" E, sample D2), and Matsudo (35◦48-06" N, 139◦58-58" E, sample D3). For comparison, a red microbial mat sample was taken from Nikko Yumoto hot spring (36◦48-30" N, 139◦25-29" E, sample H1). All samples were stored in polyethylene bottles, transported in an insulated cooler, and used for analysis immediately upon return to the laboratory. Samples used for direct cell counting were fixed in situ with ethanol (final concentration, 50% (*v*/*v*)).


**Table 1.** Physicochemical characteristics of colored bloom/mat samples studied.

