Accepted Manuscript

Review

Purification and reuse of non-point source wastewater via Myriophyllum-based

integrative biotechnology: A review

Feng Liu, Shunan Zhang, Pei Luo, Xuliang Zhuang, Xiang Chen, Jinshui Wu

PII: S0960-8524(17)31297-X
DOI: http://dx.doi.org/10.1016/j.biortech.2017.07.181
Reference: BITE 18601

To appear in: Bioresource Technology

Received Date: 30 May 2017

Revised Date: 29 July 2017
Accepted Date: 31 July 2017

Please cite this article as: Liu, F., Zhang, S., Luo, P., Zhuang, X., Chen, X., Wu, J., Purification and reuse of non-
point source wastewater via Myriophyllum-based integrative biotechnology: A review, Bioresource Technology
(2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.07.181

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title page

Purification and reuse of non-point source wastewater via

Myriophyllum-based integrative biotechnology: A review

Feng Liua,b, Shunan Zhanga,b, Pei Luoa,b, Xuliang Zhuangc, Xiang Chena,d, Jinshui
Wua,b*

a
Key Laboratory of Agro-ecological Processes in Subtropical Regions, Institute of
Subtropical Agriculture, Chinese Academy of Sciences, Hunan 410125, P. R. China
b
Changsha Research Station for Agricultural & Environmental Monitoring, Institute of
Subtropical Agriculture, Chinese Academy of Sciences, Hunan 410125, P. R. China
c
Key Laboratory of Environmental Biotechnology, Research Center for
Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, P. R.
China
d
University of Chinese Academy of Sciences, Beijing 100039, P. R. China

*
Corresponding author at: No. 644, the Second Yuanda Road, Furong District, Changsha,
Hunan 410125, P.R. China.

Tel: +86-731-8461-5234; Fax: +86-731-8461-9736

Email: jswu@isa.ac.cn (J. Wu)

1
Abstract

In this review, the applications of Myriophyllum-based integrative biotechnology to

remove common non-point source (NPS) pollutants, such as nitrogen, phosphorus,

heavy metals, and organic pollutants (e.g., pesticides and antibiotics) are summarized.

The removal of these pollutants via various mechanisms, including uptake by plant and

microbial communities in macrophyte-based treatment systems are discussed. This

review highlights the potential use of Myriophyllum biomass to produce animal feed,

fertilizer, and other valuable by-products, which can yield cost-effective returns and

attract more attention to the regulation and recycling of NPS pollutants. In addition, it

demonstrates that utilization of Myriophyllum species is a promising and reliable

strategy for wastewater treatment. The future development of sustainable

Myriophyllum-based treatment systems is discussed from various perspectives.

Keywords: Biotechnology; Aquatic macrophyte; Plant assimilation; Myriophyllum

biomass; Sustainable wastewater recover

1. Introduction

Agricultural non-point source (NPS) pollution is major contributor of nitrogen (N)

and phosphorus (P) overloading in aquatic ecosystems, and it results in accelerated

eutrophication of many rivers, lakes, and estuaries worldwide (Carpenter et al., 1998).

Various technological approaches have been developed and applied to protect water

quality and control agricultural pollution. Ecological engineering technologies, which

are more economical than conventional wastewater treatment plants, have been

successfully used to treat domestic, agricultural, and industrial wastewater (Roy, 2017;

2
Vymazal, 2011). In particular, macrophyte-based wastewater treatment systems, such as

floating islands/beds, constructed wetlands, and deep flow techniques, are promising

and utilize aquatic plants as renewable resources (Gottschall et al., 2007; Hu et al., 2008;

Xu and Shen, 2011).

Aquatic plants are important for the removal of contaminants from wastewater and

nutrient reuse, as plant assimilation removes a certain proportion of N and P (Vymazal,

2007), and plant harvest and utilization provides a profitable method of recycling

nutrients (Xu and Shen, 2011). Macrophytes were first used for wastewater treatment by

Käthe Seidel in Germany, in the early 1950s, at the Max Planck Institute in Plön

(Vymazal, 2011). For application in bioremediation, aquatic plants should have high

productivity and excellent nutrient uptake ability (Hadad et al., 2006). Because of their

high biomass production and nutrimental value, species such as Eichhornia crassipes,

several Lemna species (e.g., L. gibba, L. minor, and L. trisulca), and Spirodela

polyrhiza are frequently used in sustainable wastewater treatment systems (Malik, 2007;

Xu and Shen, 2011). The harvested plant biomass can be used to generate valuable

products, such as animal feed, biofuel, green manure, and compost. Recently, edible

aquatic swamp cabbage vegetation (Ipomoea aquatica Forssk.) has been used in

effective agricultural wastewater treatment, and it generates a substantial economic

income (Hu et al., 2008; Zhang et al., 2014). Additionally, three edible aquatic

vegetables, including I. aquatica, Zizania latifolia, and Nasturtium officinale, have been

planted consecutively in a full-scale integrated constructed wetland to recycle nutrients

for advanced purification of wastewater effluent from swine production (Zhang et al.,

2017).

The genus Myriophyllum (Haloragaceae) includes commonly occurring species such

3
as M. aquaticum, M. brasiliense, M. spicatum, and M. verticillatum and has a

cosmopolitan distribution in freshwater lakes, ponds, streams, and canals (Moody and

Les, 2013). Myriophyllum, a fast-growing species, particularly in warm regions, can

reproduce vegetatively, and it has been reported to reduce nutrient concentrations

significantly in effluents discharged from a sewage treatment plant in Dandenong,

Victoria, Australia, since the early 1980s, because of its adaptability (Nuttall, 1985).

Although low root:shoot ratios of 0.09–0.16 are observed for M. aquaticum, the

relatively small root system is sufficient for absorbing all the necessary nutrients for

rapid root growth of up to 1 cm d -1 (Hussner et al., 2009). In other way, M. aquaticum

can grow well in many natural water bodies, especially in nutrient-rich water, such as

high-strength swine wastewater (Liu et al., 2016; Luo et al., 2017). Based on the fast

growth and good environmental adaptability, the use of Myriophyllum species for

various wastewater treatments has attracted great concerns. Recently,

Myriophyllum-based treatment systems are extensively reported to be beneficial to the

remediation of polluted ecosystems owing to low cost, good effectiveness, and less

maintenance requirement. However, comprehensive evaluation of development

potential of Myriophyllum-based biotechnology is scarce until date. The focus of this

paper is to review the performances and mechanisms of Myriophyllum species on

uptake and removal of nutrients, heavy metals and organic pollutants from wastewater.

The potential of Myriophyllum-based integrative biotechnology for nutrient recycling

from wastewater by utilizing plant biomass to produce valuable by-products is

highlighted. Moreover, future perspectives of developing more promising practices to

improve Myriophyllum-based treatment systems are outlined, which might boost the

development of Myriophyllum-based biotechnology for water pollution control.

4
2. Performance of Myriophyllum species on contaminant removal

2.1. N assimilation and removal

The assimilation of N by Myriophyllum not only varies among species, but also

depends on environmental conditions and physicochemical properties of wastewater,

including nitrogen forms and nitrogen loadings (Shuskey et al., 2009). Plant species and

pollutant loading rates are crucial influencing factors on the removal performance in

phytoremediation by constructed wetlands (Wu et al., 2015). Myriophyllum species can

grow in a relatively broad range of N concentrations. In low-nutrient conditions, M.

aquaticum develops adventitious roots for nutrient uptake (Wersal and Madsen, 2011),

and M. spicatum enhances N uptake by combining by roots uptake from the sediment

and stems and leaves uptake from water (Best and Mantai, 1978). Therefore, several

pathways of nutrient uptake by Myriophyllum species in nutrient-rich water can improve

removal ability. To reduce toxicity of high ammonium-nitrogen (NH4+-N)

concentrations, M. brasiliense can produce short roots and few new buds, and apply

NH4+-N assimilation and extrusion as a strategy to prevent cytoplasmic NH4+-N

accumulation in the cells (Saunkaew et al., 2011). In addition, M. aquaticum can

convert accumulated NH4+-N into nitrate nitrogen (NO3--N) to avoid NH4+-N toxicity in

plant tissues. Compared to widely used wetland plants, such as Schoenoplectus validus,

Typha latifolia, Sagittaria latifolia, Juncus effusus, and Cyperus alternifolius, M.

aquaticum can better tolerate NH4+-N concentrations exceeding 200 mg L-1 (Zhang et

al., 2017). M. brasiliense grows well in NH4+-N-enriched water with concentrations as

high as 70 mg L-1 (Saunkaew et al., 2011). Obviously, nutrient uptake by Myriophyllum

species is beneficial for treating high-strength NH4+-N wastewater (Table 1). For

5
high-strength swine wastewater treatment, M. aquaticum uptake accounts for 17.9–42.2%

of the initial total nitrogen (TN) load (Liu et al., 2016; Zhang et al., 2017). M.

aquaticum is observed to have significantly higher N accumulation than that of

Alternanthera philoxeroides, which contributes to higher TN removal (96.4%) and a

further reduction in NH3 volatilization in swine wastewater treatment (Zhang et al.,

2016). Similarly, M. aquaticum is considered as the most effective plant species for

mitigating NH4+-N and nitrate (NO3--N) from runoff in the second year, compared to T.

latifolia, Panicum hemitomon, Thalia dealbata, and Echinodorus cordifolius (Moore et

al., 2016). It is reported that approximately 90% of the increase in biomass of M.

aquaticum contributes to the removal of 88.3% of total Kjeldahl nitrogen and 93.6% of

total phosphate (TP) from polluted water (Souza et al., 2013).

2.2. P assimilation and removal

Plant harvest is an effective method of P removal from wastewater (Zhang et al.,

2014). Three macrophytes, namely M. aquaticum, Paspalum paspalodes, and

Ranunculus repens, with P removal rates of 0.043–0.086 g m-2 d-1 (as measured in

studies on P bioaccumulation in plant tissues) can be planted in floats (Wen and

Recknagel, 2002). In swine wastewater treatment, P content in harvested M. aquaticum

from surface flow-constructed wetlands are in the range of 3.5–8.9 g kg-1 dry weight,

and annual harvested P mass ranges from 15.1 to 40.9 g m-2 yr-1. These results are

higher than those of other wetland plants, namely, Cyperus papyrus (5.0 g m-2 yr-1),

Phragmites australis (12.0 g m-2 yr-1), T. latifolia (18.0 g m-2 yr-1), Pistia stratiotes (4.0

g m-2 yr-1), Potamogeton pectinatus (4.0 g m-2 yr-1), and Ceratophyllum demersum (1.0

g m-2 yr-1) (Luo et al., 2017). The available data show that plant uptake accounts for

3–60% of P removal, depending on plant species and nutrient loading rates (Cooke,

6
1992; Gottschall et al., 2007; Tanner et al., 1995). As in the case of N uptake, different

pathways of nutrient uptake by roots, shoots, and leaves of Myriophyllum species

increase high potential for P removal from polluted water.

Apart from interspecific differences, the capacity for P uptake by plants partly

depends on the designed operation parameters of wastewater treatment systems, such as

hydraulic retention time, loading rate, wastewater type, and local climatic condition

(Vymazal, 2007; Wu et al., 2015). The annual P uptake by plants is observed to increase

from 2.2 to 3.6 g m-2 with increasing hydraulic retention times from 2 to 5.5 days for

dairy parlor wastewater treatment (Tanner et al. 1995). Appropriate plant harvest

management strategies contribute to more pollutant removal from wastewater. Vymazal

(2007) points out that P uptake by free-floating macrophytes via harvest management is

useful for treating wastewater with light P loading (e.g., 10–20 g P m-2 yr-1). Luo et al.

(2017) reported that P uptake by M. aquaticum via multiple harvests could contribute

22.5–59.6% P removal from swine wastewater. The significant difference can be

explained by the fact that the annual harvest biomass of M. aquaticum declines from

25.2 kg m-2 in diluted swine wastewater to 4.0 kg m-2 in original swine wastewater,

thereby leading to decreased TP uptake by plants from 30.2 to 2.7 g m-2 (Luo et al.,

2017). Both the growth patterns of plants and P concentrations in plant tissue vary with

the local climate condition. Generally, P uptake by aquatic macrophytes is maximized

during the peak growing season, and it decreases or even stops in the winter. For

example, the growth of M. aquaticum is inhibited at high temperature conditions (>

35 °C) during summer and at low temperature conditions (< 5 °C) during winter (Luo et

al., 2017), which may decrease P uptake by M. aquaticum. Furthermore, an increased

number of harvests during vigorous growth periods per local seasons and climates is

7
beneficial in P removal (Zhang et al., 2014), although optimizing the harvest strategy

for P removal still requires further investigation (Luo et al., 2017; Vymazal et al., 2010).

2.3. Removal of heavy metals

Macrophytes differ in tolerance and accumulation of heavy metals (Rezania et al.,

2016). M. aquaticum has greater tolerance against heavy metals such as nickel (Ni), lead

(Pb), zinc (Zn), copper (Cu), and mercury (Hg) than that of T. latifolia, Lemna minor,

and Egeria densa (Harguinteguy et al., 2015; Romero-Hernandez et al., 2017). It

displays high tolerance against mixed metal solutions containing Cu, Pb, Hg, and Zn at

par with E. crassipes, which implies that this species can be a suitable candidate for

phytoremediation of heavy metals (Romero-Hernandez et al., 2017). It is illustrated that

metal accumulation in M. aquaticum accounts for a certain percentage of metals

removal from contaminated wastewater, which helps remediate metal-contaminated

runoff from a coal storage pile (Collins et al., 2005). The capability of Myriophyllum

spicatum for cadmium (Cd), Cu, Pb, and Zn accumulation is higher than that of Typha

domingensis and Phragmites australis, but lower than Ceratophyllum demersum and

Eichhornia crassipes (Fawzy et al., 2012). It is reported that M. spicatum efficiently

removes cobalt (Co), Ni, Cu, and Zn from wastewater, and that the pH value acts as an

important influencing factor, because high pH values lead to significant increase in the

maximum metal adsorption capacity (Wang et al., 1996). In conclusion, more effective

metal phytoremediation can be achieved by increasing plant yield, which is facilitated

by cultivation of fast-growing plant species with proper environmental conditions

(Rezania et al., 2016).

The percentage of metals in dry tissue lower than 0.1% indicates that M. aquaticum

cannot be considered as hyperaccumulator (Romero-Hernandez et al., 2017). High

8
tolerance to metal toxicity and low metal accumulation in M. aquaticum could be

explained in the as follows. Firstly, root exudates and the oxidation environment play

important roles in controlling metal precipitation in root-sediment interface zones,

which act as physical barriers for mobile metals in the sediments; and changes in

morphology of M. aquaticum with thickening in the apical roots decrease the

susceptibility to metal toxicity and root permeability to metals (Teuchies et al., 2012).

Higher rates of radial oxygen loss and root porosity of wetland plants always induce to

form more Fe/Mn plaque, which is beneficial to higher arsenic (As) tolerance and lower

As translocation factor (Li et al., 2011). Similarly, Zn is observed to significantly

increase the thickness of outer cortex and lignification in cell walls significantly to

inhibit root permeability of Aegiceras corniculatum, Bruguiera gymnorrhiza, and

Rhizophora stylosa (Cheng et al., 2010).

2.4. Removal of organic contaminants

As a green technology, phytoremediation is promising for treating hazardous

organic pollutants (Alkorta and Garbisu, 2001). Myriophyllum species have

considerable capacity for uptake and metabolism of various organic pollutants. M.

aquaticum takes up atrazine by both roots and shoots, but trifluralin and terbutryn only

by shoots and cycloxydim only by roots. The experimental data is fitted well with the

uptake model developed based on concentration factors and lipophilicity expressed as

log octanol/water partition coefficient (log Kow) of the pesticides (Turgut, 2005). The

uptake of 3-phenoxybenzoic acid by M. elatinoides shoots is rapid, accounting for 15.85%

of the total mass in water, but its roots are slower at absorbing it from sediment (Ando

et al., 2012). In addition, It was found that the uptake of phenols and its metabolites,

including 4-nitrophenol, 4-cyanophenol, 4-hydroxybenzamide, and 4-hydroxybenzoic

9
acid, dissolved in water, by shoots is approximately ten times faster than that by roots

from the sediments, and the major metabolites in M. elatinoides are glucose conjugates

(Ando et al., 2015). These findings indicate uptake of pesticides by roots and shoots of

Myriophyllum varies greatly among species.

M. aquaticum has been confirmed to have an intrinsic ability to degrade

2,4,6-trinitrotoluene (TNT) through oxidative metabolism such as the methyl oxidation

and ring hydroxylation of TNT (Bhadra et al., 1999), and reductive metabolism

evidenced by the formation of both azoxy and amine products (Wang et al., 2003). The

metabolites of 3-phenoxybenzyl alcohol, 3-(4′-hydroxyphenoxy) benzoic acid and a

monoglucose conjugate of 3-phenoxybenzoic acid detected in M. elatinoids indicate that

3-phenoxybenzoic acid is reduced, oxidized, and conjugated in the macrophyte by

corresponding enzymes, respectively, which are similar to the processes known in

terrestrial plants (Ando et al., 2012). The available data show Myriophyllum can

effectively remove organic pollutants. The decay of DDT in aqueous culture medium

planted with M. aquaticum is well predicted by first-order kinetics, and the

dehalogenation and dehydrochlorination reaction reflected by the occurrences of both

DDD and DDE contribute to short degradation half-lives of 6.3 and 7.7 hours for o,

p′-DDT and p, p′-DDT, respectively (Gao et al., 2000). The microcosms seeded with M.

sibiricum biodegrade four tetracyclines (oxytetracycline, chlortetracycline, tetracycline,

and doxycycline) fast, with a 50% dissipation time (DT50) of 1-4 days depending on

concentrations of these parent compounds; and DT50 for their degradation products were

less than 1.2 days (Sanderson et al., 2005). M. aquaticum is also reported to metabolize

oxytetracycline and tetracycline tonearly complete removal within 15 days, and its

root-secreted enzymes/metabolites play important role in fast transforming the

10
antibiotics (Gujarathi et al., 2005). Further research is necessary to better understand the

environmental behaviors, including uptake, translocation, and metabolism of various

organic pollutants in submerged-root macrophytes (Ando et al., 2015).

2.5. Allocation of pollutants in Myriophyllum

Understanding of pollutants translocation in plant tissues can inform strategic

harvesting for nutrient recovery or toxicants removal. For example, N and P content in

submerged rhizomes account for 42–89% TN and less than 20% TP of M. aquaticum,

thus harvesting shoots is an easier and more efficient method of removing P compared

to harvesting roots in the sediments (Sytsma and Anderson, 1993). Pollutant uptake by

plants usually includes two processes of movement into the aqueous phase of cells,

followed by partitioning into solid tissues (de Carvalho et al., 2007), but N and P tend to

partition in the shoots rather than in the roots of M. aquaticum in the same manner as in

E. crassipes and P. stratiotes (Polomski et al., 2009). Pb, Cu, and Zn are accumulated by

roots of Myriophyllum spicatum, but only Pb is translocated to the leaves (Fawzy et al.,

2012). A similar study shows that the differences of metal allocation to the tissues of

Myriophyllum species result in significantly higher Ni, Zn, and Fe concentrations in the

roots of M. aquaticum than that in the stems (Teuchies et al., 2012). The movement and

partition of organic pollutants in roots and shoots of Myriophyllum species is similar to

that of metals; i.e., the translocation of herbicides (triclopyr, fluridone, and penoxsulam)

from shoots to roots is extremely limited with less than 3% of the total mass absorbed

by M. spicatum roots (Vassios, 2012).

3. Microorganisms in Myriophyllum wastewater treatment systems

3.1. Effects of plants on microbial distribution

11
 Aquatic macrophytes supply surface and substrate for microbial growth, and have

strong linkage with microbial biomass, diversity, and functional activity. These factors

play important roles in the mineralization and biogeochemical transformation processes

of pollutants (Pang et al., 2016; Stottmeister et al., 2003). Particularly in the rhizosphere,

radial oxygen loss of aquatic plants improves oxygen conditions in the water-flooded

environment, and stimulates aerobic microbial activity for high removal efficiency of

nutrients and organic pollutants (Braeckevelt et al., 2007; Stottmeister et al., 2003). The

oxic conditions caused by the roots of M. aquaticum favor the growth of

ammonia-oxidizing bacteria (AOB) and nitrifying organisms, which ensure consistent

year-round removal of N in a tertiary treatment system (Zhang et al., 2017; Nuttall,

1985). The optimal oxygen concentration for complete nitrification is greater than 2 mg

L-1 (Princic et al., 1998). M. aquaticum enhances the dissolved oxygen (DO)

concentration to 3.7 mg L-1 in wetland microcosms, which is higher than the 1.9 mg L-1

dissolved oxygen concentration obtained by using A. philoxeroides. These result in

faster nitrification rates and ammonia removal from swine wastewater in M. aquaticum

wetland microcosm (Zhang et al., 2016). However, the anoxic conditions of the flooded

benthos containing decaying litter material is conducive for anaerobic

processes—denitrification, anammox, sulfate reduction, and/or methanogenesis (Hadad

et al., 2006; Stottmeister et al., 2003). Microbial N transformation involves many N

conversion pathways and energy generation and consumption for wastewater treatment

(Table 2). Ammonium oxidizers obtain more energy (275 kJ mol-1) than nitrite oxidizers

(74 kJ mol-1) from nitrogen oxidation, which promote synthesis and growth of

microorganisms. Wetland plants may facilitate microbial denitrification by providing

organic carbon via root exudates (Zhai et al., 2013). Significantly, the co-occurrences of

12
aerobic and anaerobic environments implement the coupled nitrification and

denitrification processes for improving the transformation of N pollutants into N 2 and

N2O (Lee et al., 2009). Coupled nitrification and denitrification account for about 60%

TN removal from swine wastewater (Liu et al., 2016). It has been reported that M.

aquaticum enhances simultaneous growth of nitrifying and denitrifying bacteria (Souza

et al., 2013). Collins et al. (2004) observed that bacterial assemblages on M. aquaticum

are significantly different from that of other wetland plants, and suggested that

plant-microbial interactions can be species-specific.

Recently, insights into microbial aggregates with high stability and bacterial density,

developed on solid substrata in natural aquatic ecosystems, indicate their potential

application to remove various pollutants (Wu et al., 2012). Compared to floating plants,

submerged macrophytes with complex architecture can provide greater accessible

surface area for epiphytic microorganisms and further improve bacterial density,

diversity, and quality of microbial aggregates in macrophyte-biofilm systems (Cattaneo

et al., 1998). Under this framework, the density of microbes in biofilms on leaves of M.

verticillatum is higher than those of the floating macrophytes Nymphoides peltata and

Trapa japonica. Nitrosospira and Photobacter are the primary nitrifiers and denitrifiers

in these biofilms, respectively (Pang et al., 2016).

Generally, microbial activities are extremely sensitive to pH. For example, the

optimal pH ranges for nitrification and denitrification activities are 7.0–8.0 and 6.0–8.0,

respectively (Vymazal, 2007). Aquatic macrophytes can utilize N and lower pH during

respiration and litter decomposition (Collins et al., 2004). Thus, lower pH provides

undesirable conditions for precipitation of metals (Collins et al., 2004). Metal

availability and mobility may be altered by plant exudates and microbes.

13
3.2. Significance of microbial communities in macrophyte-based systems for wastewater

treatment

Exploration of the kinetics and interactions between microbial communities and

emergent plants is important to understand the nitrogen transformation mechanisms and

maintain consistent N removal efficiency (Vymazal, 2007; Wu et al., 2015).

Microorganisms are well known to be responsible for the transformation of most N, P,

and organic compounds, but further exploration of the significance of microbial

communities in macrophyte-based treatment systems, including constructed wetlands,

are needed (Truu et al., 2009). The aerobic conditions in the plant rhizosphere promote

formation of sulfur-oxidizing bacteria, which participate in oxidation of sulfides to

sulfites, and subsequently to sulfates. Under reducing conditions, sulfates are reduced to

sulfites and sulfides by sulfate-reducing bacteria. The sulfides may form insoluble

precipitates by reacting with heavy metals such as Cu, Zn, and Ni. Sulfate reduction has

been recognized as an important mechanism for metals removal in constructed wetlands

(Stein et al., 2007; Teuchies et al., 2012). In a purification system planted with M.

elatinoides for swine wastewater treatment, AOB ammonia monooxygenase α-subunit A

(amoA) gene copy numbers in the sediments are significantly higher (p < 0.05) than

ammonia oxidizing archaeal (AOA) amoA copy numbers, and the denitrification

functional genes of nirK had higher abundance than nirS. These microbial community

distributions occur in response to the main environmental factors of DO and NO3--N (Li

et al., 2015a; Li et al., 2015b). In a full-scale integrated constructed wetland, AOA

amoA and narG gene copy numbers in sediments are significantly higher in a wetland

unit vegetated with M. aquaticum than those with I. aquatica, Z. latifolia, and N.

officinale (p < 0.05), and further analysis demonstrated that ammonia oxidizer and

14
denitrifier distributions are affected by environmental factors of wetland plants and

concentrations of NH4+-N and NO3--N (Zhang et al., 2017). The bacteria performing

anaerobic ammonium oxidation (anammox) are abundant, with 7.5 × 105–3.5 × 106

copies of the marker gene hzsB g-1 dry sediment in a pilot-scale surface-flow M.

aquaticum wetland, and the presence of diverse anammox bacteria indicate that the

anammox process can be a promising N removal pathway for swine wastewater

treatment (Chen et al., 2017).

4. Biomass and potential utilization

4.1. Plant growth and biomass production

Plant species with fast growth are desirable for macrophyte-based wastewater

treatment systems. M. aquaticum in the floats have growth rates of 1.77–7.12 g m-2 d-1

of dry matter (dm), when grown in agricultural drainage waters (Wen and Recknagel,

2002). The vigorous growth of M. aquaticum in wastewater is inconsistent, as indicated

by hydraulic retention times of 6 and 12 days according to different growth rates of 2.11

and 0.91 g dm m-2 d-1, respectively (Snow and Ghaly, 2008). Compared to growth rates

in the range of 15.22–39.45 g dm m-2 d-1 for E. crassipes (Gutiérrez et al., 2001), which

is regarded as one of the most productive plants on earth (Malik, 2007), lower growth

rates of 1.77–7.12 g dm m-2 are reported for Myriophyllum species (Wen and Recknagel,

2002). However, based on the vigorous growth of M. aquaticum in swine wastewater, a

multiple harvest scheme can result in high harvested biomass in the range of 5.34–13.42

g dm m-2 d-1 (Luo et al., 2017). Obviously, plants with fast growth will quickly cover

water bodies, limiting further growth; therefore, continuous harvesting is needed to

maintain an optimum plant density (Mwale and Gwaze, 2013).

15
4.2. Potential utilization of Myriophyllum

Aquatic plants have been used as a source for production of animal feed, green

matter and compost, biosorbent, biofuels, and even human food (Malik, 2007; Yap et al.,

1979). Better utilization of massive Myriophyllum biomass as valuable by-products

would provide significant economic benefits to offset the labor costs of its harvest. M.

aquaticum in particular has been proposed for processing into animal feed and organic

fertilizer (Luo et al., 2017).

4.2.1. Animal feed

The feasibility of using aquatic plants for feed ingredients depends on their biomass

and nutritive value. As the most limiting nutrient in feed systems, the supply of

sufficient protein is a significant issue in utilizing forage plants as alternative

ingredients (Leterme et al., 2005). Compared to other eight aquatic plants such as E.

crassipes, Canna indica, Hydrocotyle verticillata and so on, M. aquaticum is reported to

have a higher crude protein content of 25.5% (Zhao et al., 2012). This value is slightly

lower than 19.8–45.0% reported in duckweed (El-Shafai et al., 2007; Leng et al., 1995),

but much higher than 8.1% and 8.0% recorded in corn and rice, respectively (Sauvant et

al., 2016). Various studies have demonstrated the successful nutritional potential of E.

crassipes and duckweed as animal feed (Lu et al., 2008; Rusoff et al., 1980). A

comparison of these results indicates that M. aquaticum is sufficiently rich in protein to

be considered for feed development for livestock and poultry. Furthermore, protein

quality, a reflection of how closely the amino acid content in plant tissues match the

needs of farm animals (Cheng and Stomp, 2009), is an important indicator for

evaluating the suitability of forage plants as animal feed. M. aquaticum has much higher

levels of eleven key amino acids than that of corn and rice, and even soybeans (except

16
cystine and methionine) (Table 3). In particular, the provision of below-average crude

protein content but greater content of ten essential amino acids by M. aquaticum in

comparison to that in duckweed highlights that M. aquaticum could contain more

high-quality protein. Considering essential mineral elements, the levels of Ca, Mg, Fe,

and Mn in M. aquaticum can meet the dietary requirements of poultry and livestock

(Zhao et al., 2012), and meet the daily Ca and Mg requirements of shellfish and fish

(Snow and Ghaly, 2008).

Several studies have reported the successful use of Myriophyllum species as animal

fodder. Compared to a basal diet, two diets with 10% and 20% of corn replaced by a

corresponding weight of mixed aquatic plant matter, including 25% M. spicatum,

showed no negative effect on the growth rate of pigs (Yap et al., 1979). Furthermore,

pigs fed on 10% aquatic plant ration showed significantly improved feed intake

efficiency (Yap et al., 1979). As forage for fish, the herbivorous grass carp

(Ctenopharyngodon idella) preferred fresh M. aquaticum and M. spicatum to E.

crassipes (Pine and Anderson, 1991). These findings indicate that Myriophyllum species

can be strong alternative ingredients for livestock feed.

4.2.2. Organic fertilizer

Surface application of harvested aquatic plants as mulch is a good practice in

agricultural cultivation. In subtropical China, tea plantations mulched with M.

verticillatum had increased soil organic matter and nutrients (e.g., N, P, K) at the 0–20

cm layer, resulting in higher farm productivity and improved tea quality. Composting is

another option for the utilization of M. aquaticum as a manure resource (Thiébaut and

Dutartre, 2009). To reduce the water content of M. spicatum to acceptable levels for

compost fertilizing, a suitable mixture with several additives of peanut shells, poultry

17
litter, and tobacco paunch manure compost can be successfully composted in a high-rate

aerobic composter (Wile et al., 1978). The compost produced from M. spicatum can be

successfully applied as biosorbent for removal of Cd 2+, Ni2+, Pb2+, Cu2+, and Zn2+, but

the amount of heavy metals absorbed by M. spicatum compost is lower than other

common composts, which indicates that M. spicatum compost can be further applied as

manure for soil quality improvement after use as a low-cost biosorbent (Milojković et

al., 2014).

4.2.3. Others

Starch and other carbohydrates in dried plants can be used for biofuel production

(Xu et al., 2012). The carbohydrate content of Myriophyllum biomass is in the range

from 40.0–60.9%, which is comparable to that of water hyacinth (31.9–61.1%) and

duckweed (31.0–75.0%) (Table 3). Previous studies have demonstrated that the

utilization of E. crassipes and duckweed in biofuel production would be feasible

(Baliban et al., 2013; Nigam, 2002), suggesting that Myriophyllum might have potential

applications in this sector. The crude fiber content of 11.4–20.5% in Myriophyllum

biomass is similar to that in water hyacinth (16.3–18.9%), which has been successfully

converted to rope, fiberboard, and high-quality paper when mixed with jute or waste

paper (Malik, 2007). Overall, the potential utilization of Myriophyllum for commercial

application prospects should be investigated in future.

5. Cost-benefit analysis and social acceptance

5.1. Cost-benefit analysis

The cost-benefit of wastewater treatment project mainly involves initial investment

and operational costs, and ecological and economic benefits. For the sustainable

18
application of Myriophyllum-based integrative biotechnology, the massive

Myriophyllum biomass may be utilized as animal feed or organic fertilizer. Therefore,

the initial investment costs primarily contain two parts: (1) construction cost of

wastewater treatment systems and (2) expenditures on animal feed or organic fertilizer

production workshops and machineries. The operating costs involve land rent cost,

maintenance cost of wastewater treatment system, workers’ wage, harvesting cost of

Myriophyllum, transportation cost, and energy consumption. Similar to artificial

wetlands, Myriophyllum-based integrative biotechnology is reported to have lower

construction and operational costs than conventional wastewater treatment plants

(WWTPs) (Liu et al., 2016; Zhang et al., 2017). Besides the non-market benefits of

water quality improvement, the conversion of Myriophyllum biomass to valuable

products of animal feed and organic fertilizer contribute to the additional benefits that

could be an important driving force for widespread application of Myriophyllum-based

biotechnology.

5.2. Social acceptance

Phytoremediation, as a cost-effective and environment-friendly remediation

technology with direct use of a wide range of plant species to removal and degrade

pollutants has been widely accepted (Rezania et al., 2016). To date, the

Myriophyllum-based treatment systems have been successfully constructed to treat

many kinds of wastewater such as swine wastewater, runoff from farmland, secondary

effluents from WWTPs (Zhu and Chen, 2014, Liu et al., 2013, Zhang et al., 2017). In

light of the above, economic feasibility analysis further ensures the development of

Myriophyllum-based biotechnology, which is likely to emerge as a sustainable method

and potential alternative for wastewater treatment.

19
6. Future perspectives

Utilization of aquatic macrophytes for wastewater treatment is promising and

reliable, with the distinct advantage that the large amounts of N and P assimilated by

plants can be ultimately removed from the wastewater during harvest. The present

review has mainly focused on the recent findings on Myriophyllum species for the

removal of nutrients and metal and organic pollutants from wastewater, as well as the

potential for the production of by-product. Based on the complex formation processes

and composition of non-point-source wastewater, the treatment technologies with

low-cost and high efficiency have significant implications for aquatic environmental

remediation, particularly in the rural areas of developing regions. For this purpose,

Myriophyllum species are a reasonable option for wastewater treatment, and are

attracting due attention of more and more researchers. However, the sustainability of

macrophyte-based technology still requires further systematic study for the development

of a novel integrative biotechnology in future (Fig. 1). In summary:

(1) The review on pollutant assimilation and removal indicates that Myriophyllum

species are tolerant of high nutrient loading and have high productivity, which enables

rapid nutrient uptake. As it is known that high ammonia levels are toxic to plants, there

is need for further investigation into the ammonia-tolerance conferring physiological

mechanisms of Myriophyllum species. Although the tolerance of Myriophyllum species

to ammonia concentrations ranges up to 400 mg L-1, reliable pre-treatment technologies

are needed to control higher-strength wastewater, such as swine wastewater. To illustrate

the uptake of nutrients and other pollutants by Myriophyllum species further, the uptake

kinetic parameters and molecular mechanisms in microscopic processes should be

20
explored in depth. The joint use of C. demersum and M. verticillatum results in

enhanced nutrient removal efficiency compared to the use of these species in isolation

(Dai et al., 2014). Therefore, attention should be paid for the development of ideal

polyculture combinations of macrophyte species in macrophyte-based treatment

systems.

(2) Our review shows that the Myriophyllum-based wastewater treatment systems

are efficient for the removal of various contaminants from wastewater. Particularly,

Myriophyllum- wetlands are reported to perform well in high-strength swine wastewater

treatment. To reduce the land area requirements for constructing wastewater treatment

systems, which is the main barrier for expanding the application of plant-based

treatment technology, further strategies corresponding to higher pollutant removal

efficiency should be taken into consideration. Given the increasingly strict water quality

standards for wastewater treatment, the addition of optimal design, operating parameters,

and novel enhancement technologies (e.g., effluent recirculation, microbial

augmentation, and artificial rapid infiltration systems) into macrophyte-based treatment

systems will require extensive investigation in future.

(3) Aquatic macrophytes are essential for the establishment and development of

distinct communities of bacteria in biofilms attached to leaves, stems, and roots (known

as epiphytic microbes). This review focuses on the function and structure of bacterial

communities in the rhizosphere that are directly affected release of oxygen and exudates

by roots, and the surrounding water quality, all of which have important roles in

biotransformation processes for pollutant removal. However, the complex interactions

between Myriophyllum species and epiphytic microbes are not well understood. In order

to provide a useful basis for enhancing remediation of contaminated environments,

21
further exploration of synergistic action by both Myriophyllum species and

microorganisms is necessary.

(4) Optimal harvest of Myriophyllum species is crucial to achieve sustainable

performance, and large-scale utilization of the harvested biomass is an important motive

force for continuous harvesting and biomass disposal. This review indicates that

Myriophyllum species have been used to produce animal fodder and compost, but the

net profit generated by the practice of biomass resource recovery requires further

economic accounting. Obviously, an appropriate harvest strategy to maximize biomass

recycling would be beneficial in the successful recovery of nutrients from wastewater.

Otherwise, the quality and safety of feed materials are given priority to the production

of animal feed. In order to increase economic profit further, the exploration of purified

water resources in Myriophyllum–based treatment systems for aquaculture can be

feasible. Overall, there is a need to establish technical and commercial patterns of such

applications to achieve the recycling and utilization of resources in regulating non-point

source pollution.

7. Conclusions

Development of low-cost biotechnology with resource recovery is an urgent need

for controlling non-point source pollution especially in rural areas. This review indicates

that plant uptake and microbial community associated with Myriophyllum greatly

contribute to removal of nutrient, metal, and organic pollutants from wastewater. The

conversion of Myriophyllum biomass to valuable outcomes such as animal feeds and

compost provides insight into the achievable sustainability of wastewater treatment

22
technology. Surely, optimized integration of nutrient recycling from wastewater and

efficient processing of biomass with high economic return is required to promote

widespread implementation of Myriophyllum-based biotechnology in future.

Acknowledgments
This study was financially supported by the Youth Innovation Team Project of ISA,

CAS (2017QNCXTD_LF), the Key Research Project of Frontier Science of Chinese

Academy of Sciences (QYZDJ-SSW-DQC041), the National Key Research and

Development Program (2017YFD0800104), the National Science and Technology

Supporting Project (2014BAD14B0104), and the National Natural Science Foundation

of China (41601273).

References
1. Abdelhamid, A.M., Gabr, A.A., 1991. Evaluation of water hyacinth as feed for
ruminants. Arch. Anim. Nutr. 41, 745–756.
2. Alkorta, I., Garbisu, C., 2001. Phytoremediation of organic contaminants in soils.
Bioresour. Technol. 79, 273–276.
3. Ando, D., Fujisawa, T., Katagi, T., 2012. Uptake, translocation, and metabolism of
3-phenoxybenzoic acid in the submerged rooted macrophyte water milfoil
(Myriophyllum elatinoides). J. Pestic. Sci. 37, 342–346.
4. Ando, D., Fujisawa, T., Katagi, T., 2015. Uptake, translocation, and metabolism of
phenols by submerged rooted macrophyte, water milfoil (Myriophyllum elatinoides).
J. Agr. Food Chem. 63, 5189–5195.
5. Baliban, R.C., Elia, J.A., Floudas, C.A., Xiao, X., Zhang, Z., Li, J., Cao, H., Ma, J.,

23
 Qiao, Y., Hu, X., 2013. Thermochemical conversion of duckweed biomass to
gasoline, diesel, and jet fuel: process synthesis and global optimization. Ind. Eng.
Chem. Res. 52, 11436–11450.
6. Best, M.D., Mantai, K.E., 1978. Growth of Myriophyllum: Sediment or lake water
as the source of nitrogen and phosphorus. Ecology 59, 1075–1080.
7. Bhadra, R., Spanggord, R.J., Wayment, D.G., Hughes, J.B., Shanks, J.V., 1999.
Characterization of oxidation products of TNT metabolism in aquatic
phytoremediation systems of Myriophyllum aquaticum. Environ. Sci. Technol. 33,
3354–3361.
8. Braeckevelt, M., Rokadia, H., Imfeld, G., Stelzer, N., Paschke, H., Kuschk, P.,
Kästner, M., Richnow, H., Weber, S., 2007. Assessment of in situ biodegradation of
monochlorobenzene in contaminated groundwater treated in a constructed wetland.
Environ. Pollut. 148, 428–437.
9. Carpenter, S.R., Caraco, N.F., Correll, D.L., Howarth, R.W., Sharpley, A.N, Smith,
V.H., 1998. Nonpoint pollution of surface waters with phosphorus and nitrogen.
Ecol. Appl. 8, 559–568.
10. Cattaneo, A., Galanti, G., Gentinetta, S., Romo, A.S., 1998. Epiphytic algae and
macroinvertebrates on submerged and floating-leaved macrophytes in an Italian
lake. Freshwater Biol. 39, 725–740.
11. Cervantes, F.J., Cuervo-López, F., Hernández, J.G., 2016. Fundamentals of the
biological processes for nitrogen removal. Technologies for the Treatment and
Recovery of Nutrients from Industrial Wastewater, pp.112–146.
12. Chen, L., Liu, F., Jia, F., Hu, Y.J., Lai, C., Li, X., Luo, P., Xiao, R.L., Li, Y., Wu,
J.S., 2017. Anaerobic ammonium oxidation in sediments of surface flow
constructed wetlands treating swine wastewater. Appl. Microbiol. Biotechnol. 101,
1301–1311.
13. Cheng, H., Liu, Y., Tam, N. F. Y., Wang, X., Li, S. Y., Chen, G. Z., Ye, Z. H., 2010.
The role of radial oxygen loss and root anatomy on zinc uptake and tolerance in
mangrove seedlings. Environ. Pollut. 158, 1189–1196.
14. Cheng, J.J., Stomp, A.M., 2009. Growing duckweed to recover nutrients from
wastewaters and for production of fuel ethanol and animal feed. Clean – Soil Air
Water 37, 17–26.
15. Collins, B., McArthur, J.V., Sharitz, R.R., 2004. Plant effects on microbial
assemblages and remediation of acidic coal pile runoff in mesocosm treatment
wetlands. Ecol. Eng. 23, 107–115.
16. Collins, B., Sharitz, R.R., Coughlin, D.P., 2005. Elemental composition of native
wetland plants in constructed mesocosm treatment wetlands. Bioresour. Technol. 96,
937–948.
17. Cooke, J.G., 1992. Phosphorus removal process in a wetland after a decade of
receiving sewage effluent. J. Environ. Qual. 21, 733–739.
18. Dai, Y., Tang, H., Chang, J., Wu, Z., Liang, W., 2014. What’s better, Ceratophyllum
demersum L. or Myriophyllum verticillatum L., individual or combined? Ecol. Eng.
70, 397–401.

24
19. de Carvalho, R.F., Bromilow, R.H., Greenwood, R., 2007. Uptake and translocation
of non-ionised pesticides in the emergent aquatic plant parrot feather Myriophyllum
aquaticum. Pest Manag. Sci. 63, 798–802.
20. Dewanji, A., 1993. Amino acid composition of leaf protein extracted from some
aquatic weeds. J. Agr. Food Chem. 41, 1232–1236.
21. El-Shafai, S.A., El-Gohary, F.A., Nasr, F.A., Van der Steen, N.P., Gijzen, H.J., 2007.
Nutrient recovery from domestic wastewater using a UASB-duckweed ponds
system. Bioresour. Technol. 98, 798–807.
22. Fawzy, M.A., Badr, N.E.-s., El-Khatib, A., Abo-El-Kassem, A., 2012. Heavy metal
biomonitoring and phytoremediation potentialities of aquatic macrophytes in River
Nile. Environ. Monit. Assess. 184, 1753–1771.
23. Gao, J., Garrison, A.W., Hoehamer, C., Mazur, C.S., Wolfe, N.L., 2000. Uptake and
phytotransformation of o,p‘-DDT and p,p‘-DDT by axenically cultivated aquatic
plants. J. Agr. Food Chem. 48, 6121–6127.
24. Gottschall, N., Boutin, C., Crolla, A., Kinsley, C., Champagne, P., 2007. The role of
plants in the removal of nutrients at a constructed wetland treating agricultural
(dairy) wastewater, Ontario, Canada. Ecol. Eng. 29, 154–163.
25. Gujarathi, N.P., Haney, B.J., Linden, J.C., 2005. Phytoremediation potential of
Myriophyllum aquaticum and Pistia stratiotes to modify antibiotic growth
promoters, tetracycline, and oxytetracycline, in aqueous wastewater systems. Int. J.
Phytoremediat. 7, 99–112.
26. Gutiérrez, E.L., Ruiz, E.F., Uribe, E.G., Martínez, J.M., 2001. Biomass and
Productivity of water hyacinth and their application in control programs. In: Julien,
M.H., Hill, M.P., Center, T.D., Jianqing, D. (Eds), Biological and Integrated Control
of Water Hyacinth, Eichhornia crassipes. Canberra, Australia: Australian Centre for
International Agricultural Research, pp.109–120.
27. Hadad, H.R., Maine, M.A., Bonetto, C.A., 2006. Macrophyte growth in a
pilot-scale constructed wetland for industrial wastewater treatment. Chemosphere
63, 1744–1753.
28. Harguinteguy, C.A., Pignata, M.L., Fernández-Cirelli, A., 2015. Nickel, lead and
zinc accumulation and performance in relation to their use in phytoremediation of
macrophytes Myriophyllum aquaticum and Egeria densa. Ecol. Eng. 82, 512–516.
29. Hu, M.H., Ao, Y.S., Yang, X.E., Li, T.Q., 2008. Treating eutrophic water for
nutrient reduction using an aquatic macrophyte (Ipomoea aquatica Forsskal) in a
deep flow technique system. Agr. Water Manage. 95, 607–615.
30. Hussner, A., Meyer, C., Busch, J., 2009. The influence of water level and nutrient
availability on growth and root system development of Myriophyllum aquaticum.
Weed Res. 49, 73–80.
31. Janssen, M.A., Walker, K.F., 1999. Processing of riparian and wetland plant litter in
the River Murray, South Australia. Hydrobiologia 411, 53–64.
32. Juliano, B.O., 1985. Rice chemistry and technology. American Association of
Cereal Chemists, St Paul, MN, pp.757.

25
33. Karr-Lilienthal, L., Kadzere, C.T., Grieshop, C.M., Jr, G.C.F., 2005. Chemical and
nutritional properties of soybean carbohydrates as related to nonruminants: A
review. Livest. Prod. Sci. 97, 1–12.
34. Lee, C.G., Fletcher, T.D., Sun, G.Z., 2009. Nitrogen removal in constructed wetland
systems. Eng. Life Sci. 9, 11–22.
35. Leng, R.A., Stambolie, J.H., Bell, R., 1995. Duckweed – A potential high-protein
feed resource for domestic animals and fish. Livest. Res. Rural Devel. 7, 36.
36. Leterme, P., Londoño, A.M., Estrada, F., Souffrant, W.B., Buldgen, A., 2005.
Chemical composition, nutritive value and voluntary intake of tropical tree foliage
and cocoyam in pigs. J. Sci. Food Agr. 85, 1725–1732.
37. Li, H., Ye, Z.H., Wei, Z.J., Wong, M.H., 2011. Root porosity and radial oxygen loss
related to arsenic tolerance and uptake in wetland plants. Environ. Pollut. 159,
30–37.
38. Li, X., Zhang, M., Liu, F., Wu, J., Li, Y., He, Y., Zhang, S., 2015a. The significance
of Myriophyllum elatinoides for swine wastewater treatment: abundance and
community structure of ammonia-oxidizing microorganisms in sediments. PloS
One 10, e0139778.
39. Li, X., Zhang, M., Liu, F., Wu, J.., Li, Y., He, Y., Zhang, S., 2015b. Abundance and
distribution of microorganisms involved in denitrification in sediments of a
Myriophyllum elatinoides purification system for treating swine wastewater.
Environ. Sci. Pollut. Res. 22, 17906–17916.
40. Liu, F., Xiao, R., Wang, Y., Li, Y., Luo, Q., Wu, J., 2013. Effect of a novel
constructed drainage ditch on the phosphorus sorption capacity of ditch soils in an
agricultural headwater catchment in subtropical central China. Ecol. Eng. 58,
69–76.
41. Liu, F., Zhang, S., Wang, Y., Li, Y., Xiao, R., Li, H., He, Y., Zhang, M., Wang, D.,
Li, X., Wu, J., 2016. Nitrogen removal and mass balance in newly-formed
Myriophyllum aquaticum mesocosm during a single 28-day incubation with swine
wastewater treatment. J. Environ. Manage. 166, 596–604.
42. Lu, J., Fu, Z., Yin, Z., 2008. Performance of a water hyacinth (Eichhornia crassipes)
system in the treatment of wastewater from a duck farm and the effects of using
water hyacinth as duck feed. J. Envion. Sci. 20, 513–519.
43. Luo, P., Liu, F., Liu, X., Wu, X., Yao, R., Chen, L., Li, X., Xiao, R., Wu, J., 2017.
Phosphorus removal from lagoon-pretreated swine wastewater by pilot-scale
surface flow constructed wetlands planted with Myriophyllum aquaticum. Sci. Total
Environ. 576, 490–497.
44. Malik, A., 2007. Environmental challenge vis a vis opportunity: The case of water
hyacinth. Environ. Int. 33, 122–138.
45. Milojković, J.V., Stojanović, M.D., Mihajlović, M.L., Lopičić, Z.R., Petrović, M.S.,
Šoštarić, T.D., Ristić, M.D., 2014. Compost of aquatic weed Myriophyllum
spicatum as low-cost biosorbent for selected heavy metal ions. Water Air Soil Pollu.
225, 1927.
46. Moody, M.L., Les, D.H., 2013. Systematics of the aqutic angiosperm genus

26
 Myriophyllum (Haloragaceae). Syst. Bot. 35, 121–139.
47. Moore, M.T., Locke, M.A., Kröger, R., 2016. Using aquatic vegetation to remediate
nitrate, ammonium, and soluble reactive phosphorus in simulated runoff.
Chemosphere 160, 149–154.
48. Mwale, M., Gwaze, F.R., 2013. Characteristics of duckweed and its potential as
feed source for chickens reared for meat production: A review. Sci. Res. Essays 8,
689–697.
49. Nigam, J.N., 2002. Bioconversion of water hyacinth (Eichhornia crassipes)
hemi-cellulose acid hydrolysate to motor fuel ethanol by xylose-fermenting yeast. J.
Biotechnol. 97, 107–116.
50. Nuttall, P.M., 1985. Uptake of phosphorus and nitrogen by Myriophyllum
aquaticum (Velloza) Verd. Growing in a wastewater treatment system. Mar.
Freshwater Res. 36, 493–507.
51. Pang, S., Zhang, S., Lv, X.Y., Han, B., Liu, K., Qiu, C., Wang, C., Wang, P., Toland,
H., He, Z., 2016. Characterization of bacterial community in biofilm and sediments
of wetlands dominated by aquatic macrophytes. Ecol. Eng. 97, 242–250.
52. Patel, V., Desai, M., Madamwar, D., 1993. Thermochemical pretreatment of water
hyacinth for improved biomethanation. Appl. Biochem. Biotech. 42, 67–74.
53. Pine, R.T., Anderson, L.W.J., 1991. Plant preferences of triploid grass carp. J. Aquat.
Plant Manage. 29, 80–82.
54. Polomski, R.F., Taylor, M.D., Bielenberg, D.G., Bridges, W.C., Klaine, S.J.,
Whitwell, T., 2009. Nitrogen and phosphorus remediation by three floating aquatic
macrophytes in greenhouse-based laboratory-scale subsurface constructed wetlands.
Water Air Soil Pollut. 197, 223–232.
55. Princic, A., Mahne, I., Megusar, F., Paul, E.A., and Tiedje, J.M., 1998. Effects of
pH and oxygen and ammonium concentrations on the community structure of
nitrifying bacteria from wastewater. Appl. Environ. Microbiol. 64, 3584–3590.
56. Reid, M.S., Bieleski, R.L., 1970. Response of Spirodela oligorrhiza-M to
phosphorus deficiency. Plant Physiol. 46, 609–613.
57. Rezania, S., Taib, S.M., Din, M.F.M., Dahalan, F.A., Kamyab, H., 2016.
Comprehensive review on phytotechnology: Heavy metals removal by diverse
aquatic plants species from wastewater. J. Hazard. Mater. 318, 587–599.
58. Romero-Hernández, J.A., Amaya-Chávez, A., Balderas-Hernández, P., Roa-Morales,
G., González-Rivas, N., Balderas-Plata, M.Á., 2017. Tolerance and
hyperaccumulation of a mixture of heavy metals (Cu, Pb, Hg, and Zn) by four
aquatic macrophytes. Int. J. Phytoremediat. 19, 239–245.
59. Roy, E.D., 2017. Phosphorus recovery and recycling with ecological engineering: A
review. Ecol. Eng. 98, 213–227.
60. Rusoff, L.L., Blakney, E.W.Jr., Culley, D.D.Jr., 1980. Duckweeds (Lemnacae
Family): a potential source of protein and amino acids. J. Agr. Food Chem. 28,
848–850.
61. Sanderson, H., Ingerslev, F., Brain, R.A., Halling-Sørensen, B., Bestari, J.K.,

27
 Wilson, C.J., Johnson, D.J., Solomon, K.R., 2005. Dissipation of oxytetracycline,
chlortetracycline, tetracycline and doxycycline using HPLC–UV and LC/MS/MS
under aquatic semi-field microcosm conditions. Chemosphere 60, 619–629.
62. Saunkaew, P., Wangpakapattanawong, P., Jampeetong, A., 2011. Growth,
morphology, ammonium uptake and nutrient allocation of Myriophyllum brasiliense
Cambess. under high NH4+ concentrations. Ecotoxicology 20, 2011–2018.
63. Sauvant, D., Perez, J.M., Tran, G., 2004. Tables of composition and nutritional
value of feed materials: Pigs, poultry, cattle, sheep, goats, rabbits, horses, fish.
Wageningen Academic Publishers, Wageningen, the Netherlands.
64. Schmidt, I., Sliekers, O., Schmid, M., Bock, E., Fuerst, J., Kuenen, J.G., Jetten,
M.S.M., Strous, M., 2003. New concepts of microbial treatment processes for the
nitrogen removal in wastewater. FEMS Microbiol. Rev. 27, 481–492.
65. Shuskey, T., Bosch, I., Ryczek, C., Cohen, B., 2009. In situ experimental studies of
Eurasian Watermilfoil (Myriophyllum spicatum) downstream from agricultural
watersheds: Nutrient loading, foliar uptake, and growth. J. Great Lakes Res. 35,
109–116.
66. Snow, A.M., Ghaly, A.E., 2008. Assessment of hydroponically grown macrophytes
for their suitability as fish feed. Am. J. Biochem. Biotechnol. 4, 43–56.
67. Souza, F.A., Dziedzic, M., Cubas, S.A., Maranho, L.T., 2013. Restoration of
polluted waters by phytoremediation using Myriophyllum aquaticum (Vell.) Verdc.,
Haloragaceae. J. Environ. Manage. 120, 5–9.
68. Stein, O.R., Borden-Stewart, D.J., Hook, P.B., Jones, W.L., 2007. Seasonal
influence on sulfate reduction and zinc sequestration in subsurface treatment
wetlands. Water Res. 41, 3440–3448.
69. Stottmeister, U., Wiessner, A., Kuschk, P., Kappelmeyer, U., Kästner M., Bederski,
O., Müller, R.A., Moormann, H., 2003. Effects of plants and microorganisms in
constructed wetlands for wastewater treatment. Biotechnol. Adv. 22, 93–117.
70. Sytsma, M.D., Anderson, L.W.J., 1993. Biomass, nitrogen, and phosphorus
allocation in parrot feather (Myriophyllum aquaticum). J. Aquat. Plant Manage. 31,
244–248.
71. Tanner, C.C., Clayton, J.S., Upsdell, M.P., 1995. Effect of loading rate and planting
on treatment of dairy farm wastewaters in constructed wetlands—II. Removal of
nitrogen and phosphorus. Water Res. 29, 27–34.
72. Teuchies, J., De Jonge, M., Meire, P., Blust, R., Bervoets, L., 2012. Can acid
volatile sulfides (AVS) influence metal concentrations in the macrophyte
Myriophyllum aquaticum? Environ. Sci. Technol. 46, 9129–9137.
73. Thiébaut, G., Dutartre, A., 2009. Management of invasive aquatic plants in France.
In: George, H. (Ed.), Aquatic Ecosystems Research Trends. Nova Sciences
Publishers, Hauppauge, pp. 25–46.
74. Truu, M., Juhanson, J., Truu, J., 2009. Microbial biomass, activity and community
composition in constructed wetlands. Sci. Total Environ. 407, 3958–3971.
75. Turgut, C., 2005. Uptake and modeling of pesticides by roots and shoots of

28
 parrotfeather (Myriophyllum aquaticum). Environ. Sci. Pollut. Res. 12, 342–346.
76. Vassios, J.D., 2012. Herbicide absorption and translocation by Eurasian
watermilfoil and hydrilla. In: Department of Bioagricultural Sciences and Pest
Management, Doctor of Philosophy, Colorado State University.
77. Virabalin, R., Kositsup, B., Punnapayak, H., 1993. Leaf protein concentrate from
water hyacinth. J. Aquat. Plant Manage. 31, 207–209.
78. Vymazal, J., 2007. Removal of nutrients in various types of constructed wetlands.
Sci. Total Environ. 380, 48–65.
79. Vymazal, J., Kröpfelová, L., Švehla, J., Štíchová, J., 2010. Can multiple harvest of
aboveground biomass enhance removal of trace elements in constructed wetlands
receiving municipal sewage? Ecol. Eng. 36, 939–945.
80. Vymazal, J., 2011. Constructed wetlands for wastewater treatment: five decades of
experience. Envion. Sci. Technol. 45, 61–69.
81. Wang, T.C., Weissman, J.C., Ramesh, G., Varadarajan, R., Benemann, J.R., 1996.
Parameters for removal of toxic heavy metals by water milfoil (Myriophyllum
spicatum). B. Environ. Contam. Tox. 57, 779–786.
82. Wang, C., Lyon, D.Y., Hughes, J.B., Bennett, G.N., 2003. Role of hydroxylamine
intermediates in the phytotransformation of 2,4,6-Trinitrotoluene by Myriophyllum
aquaticum. Environ. Sci. Technol. 37, 3595–3600.
83. Wen, L., Recknagel, F., 2002. In situ removal of dissolved phosphorus in irrigation
drainage water by planted floats: preliminary results from growth chamber
experiment. Agr. Ecosyst. Envion. 90, 9–15.
84. Wersal, R.M., Madsen, J.D., 2011. Influences of water column nutrient loading on
growth characteristics of the invasive aquatic macrophyte Myriophyllum aquaticum
(Vell.) Verdc. Hydrobiologia 665, 93–105.
85. Wile, I., Neil, J., Lumis, G., Pos, J., 1978. Production and utilization of aquatic
plant compost. J. Aquat. Plant Manage. 16, 24–27.
86. Wu, H., Zhang, J., Ngo, H.H., Guo, W., Hu, Z., Liang, S., Fan, J., Liu, H., 2015. A
review on the sustainability of constructed wetlands for wastewater treatment:
Design and operation. Bioresour. Technol. 175, 594–601.
87. Wu, J., 2017. A systemic biological technique to treat organic, nitrogen and
phosphate polluted water. Plenary presentation at the 1st international conference on
ecotechnologies for controlling non-point source pollution and protecting aquatic
ecosystem, Nanjing, China.
88. Wu, Y., Li, T., Yang, L., 2012. Mechanisms of removing pollutants from aqueous
solutions by microorganisms and their aggregates: A review. Bioresour. Technol.
107, 10–18.
89. Xu, J., Shen, G., 2011. Growing duckweed in swine wastewater for nutrient
recovery and biomass production. Bioresour. Technol. 102, 848–853.
90. Xu, J., Zhao, H., Stomp, A.M., Cheng, J.J., 2012. The production of duckweed as a
source of biofuels. Biofuels 3, 589–601.

29
91. Yap, T.N., Pond, W.G., Wu, J.F., Stoewsand, G.S., Wszolek, P.C., Lisk, D.J., 1979.
Nutritional and toxicological studies with growing pigs fed aquatic plant rations.
Arch. Environ. Contam. Toxicol. 8, 613–619.
92. Yoshida, T., Tsubaki, S., Teramoto, Y., Azuma, J.-i., 2010. Optimization of
microwave-assisted extraction of carbohydrates from industrial waste of corn starch
production using response surface methodology. Bioresour. Technol. 101,
7820–7826.
93. Zhai, X., Piwpuan, N., Arias, C.A., Headley, T., Brix, H., 2013. Can root exudates
from emergent wetland plants fuel denitrification in subsurface flow constructed
wetland systems? Ecol. Eng. 61, 555–563.
94. Zhang, Q., Achal, V., Xu, Y., Xiang, W.N., 2014. Aquaculture wastewater quality
improvement by water spinach (Ipomoea aquatica Forsskal) floating bed and
ecological benefit assessment in ecological agriculture district. Aquacult. Eng. 60,
48–55.
95. Zhang, S., Xiao, R., Liu, F., Zhou, J., Li, H., Wu, J., 2016. Effect of vegetation on
nitrogen removal and ammonia volatilization from wetland microcosms. Ecol. Eng.
97, 363–369.
96. Zhang, S., Liu, F., Xiao, R., He, Y., Wu, J., 2017. Nitrogen removal in
Myriophyllum aquaticum wetland microcosms for swine wastewater treatment:
15
N-labelled nitrogen mass balance analysis. J. Sci. Food Agr. 97, 505–511.
97. Zhang, M., Luo, P., Liu, F., Li, H., Zhang, S., Xiao, R., Yin, L., Zhou, J., Wu, J.,
2017. Nitrogen removal and distribution of ammonia-oxidizing and denitrifying
genes in an integrated constructed wetland for swine wastewater treatment. Ecol.
Eng. 104, 30–38.
98. Zhao, F., Xi, S., Yang, X., Yang, W., Li, J., Gu, B., He, Z., 2012. Purifying
eutrophic river waters with integrated floating island systems. Ecol. Eng. 40, 53–60.
99. Zhu, S., Chen, H., 2014. The fate and risk of selected pharmaceutical and personal
care products in wastewater treatment plants and a pilot-scale multistage
constructed wetland system. Environ. Sci. Pollut. Res. 21, 1466–1479.

30
Table 1 Removal performances of nitrogen and phosphorus by different plant species.
Plant Species Wastewater type and Removal efficiency of Plant uptake mass Plant uptake References
system system contribution
Myriophyllum Swine wastewater; NH4+-N and TN: >97% TN: 5.3–7.2 g m-2 TN: 17.9–42.2% of (Liu et al., 2016;
aquaticum constructed wetland; influent N amount Zhang et al., 2017)
Myriophyllum Swine wastewater; P: 78.2–89.8% P: 15.1–40.9 g m-2 yr-1 P: 22.5–59.6% of (Luo et al., 2017)
aquaticum constructed wetland; removal
Myriophyllum Synthetic waste water; NH4+-N: 49.3%. TN: 3.25–7.75 g m-2 TN: 16–19% of the (Zhang et al., 2016)
elatinoides drainage ditches influent N amount
Pontederia Synthetic waste water; NH4+-N: 40.6% TN: 2.14–4.75 g m-2 TN: 10–12% of the (Zhang et al., 2016)
cordata. drainage ditches influent N amount
Phragmites Simulated river water; TN: 60.7%; NH4+-N: TN: 6.5 g m-2 TN: 14.3%; (Wu et al., 2011)
australis constructed wetlands 93.1%; P: 47.8% P: 0.31 g m-2 P: 10.8% of removal
Typha orientalis Simulated river water; TN: 60.9%; NH4+-N: TN: 9.6 g m-2 TN: 21.0%; P: (Wu et al., 2011)
constructed wetlands 88.3%; P: 63.2% P: 0.56 g m-2 14.3% of removal
Typha Synthetic waste water; - TN: 48–54 g m-2 yr-1 15.4–18.9% of (Maltais-Landry et
angustifolia constructed wetlands removal al. 2009)
Scirpus validus Simulated river water; TN: 66.7%; NH4+-N: TN: 22.84 g m-2 TN: 45.5%; P: (Wu et al., 2011)
constructed wetlands 89.4%; TP: 66.5% P: 1.36 g m-2 32.3% of removal
Iris pseudacorus Simulated river water; TN: 67.8%; NH4+-N: TN: 26.57 g m-2 TN: 51.9%; P: (Wu et al., 2011)
constructed wetlands 90.6%; P: 66.4% P: 1.44 g m-2 34.2% of removal

31
Table 2 Gibbs free energy of important reactions involved in nitrogen transform.

Equations ΔG0’/kJ mol-1

Nitrification

NH4+ + 1.5 O2→NO2- + H2O + 2 H+ -275a

NO2- + 0.5 O2→NO3- -74a

Denitrification

4 NO3- + 5 C + 2 H2O→2 N2 + 4 HCO3- + CO2 -492–-560b

Anaerobic ammonia oxidation

NO3- + NH4+→N2 + 2 H2 -357a

a
adopted from Schmidt et al. (2003); b adopted from Cervantes et al. (2016).

32
 Table 3 Nutritional composition of three aquatic plants and common grains.

Parameter Eichhornia Duckweed Myriophyllum Corn Rice Soybean

crassipes grain

Nutrients (% dry matter)

Carbohydrates 31.9–61.1 31.1–75.0 40.0–60.9 83.2 80 35
Crude fiber 16.3–18.9 8.8–11.0 11.4–20.5 2.2 0.5 5.2
Crude protein 11.9–20.0 19.8–45.0 22.4–25.5 8.1 8.0 34.8
Amino acid (% crude protein)
Arginine 1.1–3.6 3.8–5.3 8.0 4.7 8.2 7.4
Cystine 0.4–0.8 4.6 0.4 2.5 2.4 1.6
Histidine 0.6–1.1 1.2–2.7 4.2 2.9 2.3 2.7
Isoleucine 1.4–2.3 3.1–3.9 7.7 3.7 4.0 4.6
Leucine 2.7–5.1 5.8–9.6 11.3 12.5 7.9 7.5
Lysine 1.6–2.7 3.4–6.1 8.7 3.0 3.7 6.2
Methionine 1.3 0.8–1.4 1.4 2.1 2.3 1.5
Phenylalanine 3.4 3.6–4.5 7.3 4.9 5.1 5.0
Threonine 1.6–2.6 2.6–3.5 5.9 3.7 3.4 4.0
Tyrosine 2.2 2.2–3.1 5.3 4.2 4.6 3.5
Valine 2.0–2.8 3.5–6.8 8.1 5.0 5.9 4.8
References (1)–(4) (5)–(10) (1),(11)–(13) (14),(15) (14),(16) (14),(17)
References: (1) Snow and Ghaly (2008); (2) Abdelhamid and Gabr (1991); (3) Patel

et al. (1993); (4) Virabalin et al. (1993); (5) Xu et al. (2012); (6) Reid and Bieleski

(1970); (7) El-Shafai et al. (2007); (8) Leng et al. (1995); (9) Rusoff et al. (1980); (10)

Dewanji (1993); (11) Janssen and Walker (1999); (12) Zhao et al. (2012); (13) Wu

(2017); (14) Sauvant et al. (2016); (15) Yoshida et al. (2010); (16) Juliano (1985); (17)

Karr-Lilienthal et al. (2005).

33
Figure caption

Fig. 1. Schematic diagram of future perspectives for improving the sustainability of

Myriophyllum-based integrative biotechnology.

34
 Tolerance and uptake
(Physiological and molecular
mechanisms)

Enhancement strategies Myriophyllum-based Other economic profit

(microbial augmentation, integrative (Purified water for
effluent recirculation) biotechnology aquaculture)

Economic Biomass Nutrient

return recycling

Valuable
by-products
Fig. 1

35
Graphical abstract

Tolerance and uptake

(Physiological and molecular
mechanisms)

Enhancement strategies Myriophyllum-based Other economic profit

(microbial augmentation, integrative (Purified water for
effluent recirculation) biotechnology aquaculture)

Economic Biomass Nutrient

return recycling

Valuable
by-products

36
HIGHLIGHTS

● Contribution of uptake by Myriophyllum species to pollutants removal is analyzed.

● Myriophyllum is essential for distinct microbial community distribution.

● Myriophyllum-based biotechnology is promising for wastewater treatment and reuse.

● Valuable by-products are successfully produced by Myriophyllum biomass.

● Economic returns is proposed for sustainability of Myriophyllum treatment systems.

37