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Applied Soil Ecology 61 (2012) 171–189
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Applied Soil Ecology
journal homepage: www.elsevier.com/locate/apsoil
The potential contribution of plant growth-promoting bacteria to reduce
environmental degradation – A comprehensive evaluation
Luz E. de-Bashan a,b , Juan-Pablo Hernandez a,b , Yoav Bashan a,b,∗
a
b
Environmental Microbiology Group, Northwestern Center for Biological Research (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico
The Bashan Foundation, 3740 NW Harrison Blvd., Corvallis, OR 97330, USA
a r t i c l e
i n f o
Article history:
Received 13 June 2011
Received in revised form 19 August 2011
Accepted 4 September 2011
This study is dedicated for the memory of
the German/Spanish mycorrhizae
researcher Dr. Horst Vierheilig
(1960–2011) of CSIC, Spain.
Keywords:
Desert reforestation
Mangrove restoration
Phytoremediation
Phytostabilization
Rhizodegradation
Water bioremediation
a b s t r a c t
Plant growth-promoting bacteria (PGPB) are commonly used to improve crop yields. In addition to their
proven usefulness in agriculture, they possess potential in solving environmental problems. Some examples are highlighted. PGPB may prevent soil erosion in arid zones by improving growth of desert plants
in reforestation programs; in turn, this reduces dust pollution. PGPB supports restoration of mangrove
ecosystems that lead to improve fisheries. PGPB participate in phytoremediation techniques to decontaminate soils and waters. These include: phytodegradation, phytotransformation, bioaugmentation,
rhizodegradation, phytoextraction, phycoremediation, and phytostabilization, all leading to healthier
environments. This review describes the state-of-the-art in these fields, examples from peer-reviewed
literature, pitfalls and potentials, and proposes open questions for future research.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Plant growth-promoting bacteria (PGPB, Bashan and Holguin,
1998; PGPR, Kloepper et al., 1980) are bacterial strains isolated from
diverse environments with potential to positively influence many
parameters of plant growth and yield. As inoculants, single and
combinations of PGPB/PGPR are common and their use is increasing in agricultural practices (Díaz-Zorita and Fernández-Canigia,
2009). PGPB affect plants through a multitude of mechanisms.
Several comprehensive and critical reviews describing operational mechanisms in PGPB/PGPR were published in recent years.
It is likely that some mechanisms operating in crops are also
valid for PGPB used in studies of environmental remediation
(Bashan and de-Bashan, 2010; Lucy et al., 2004; Lugtenberg and
Kamilova, 2009). Consequently, general discussion of plausible
mechanisms for promoting plant growth will not be described in
this review.
∗ Corresponding autor at: Environmental Microbiology Group, Northwestern Center for Biological Research (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita,
La Paz, B.C.S. 23090, Mexico. Tel.: +52 612 123 8484.
E-mail address: [email protected] (Y. Bashan).
0929-1393/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsoil.2011.09.003
There is a steady and serious decline in environmental health
worldwide and impairment of many ecosystems resulting from
human activities, some on a very large scale. These include deforestation in the tropics and uncontrolled and unregulated release
of pollutants to the environment. This indirectly increases exposure to environmental contaminants, heavy metals, and excessive
nutrients (N and P) on a massive scale, impairing public health
in some countries. Traditional solutions for remediation, excavation and relocation of contaminants to landfills, are expensive and
usually impractical because of the amount of soil involved, as in
the case of mine tailings. Additionally, new contaminated sites
of extensive size continue to increase. Consequently, more costeffective remediation technologies are being investigated. One of
the emerging strategies is the use of plants to extract, mitigate,
and stabilize contaminants, which can be categorized as phytoremediation and assist in reforestation (Cunningham et al., 1995;
Ernst, 2005; Glick, 2003; Macek et al., 2000; McCutcheon and
Schnoor, 2003; McGuinness and Dowling, 2009; Mench et al., 2006;
Mukhopadhyay and Maiti, 2010; Pilon-Smits and Freeman, 2006;
Weyens et al., 2009). As a strategy, and especially in comparison to
removal and relocation of contaminants, phytoremediation is inexpensive. Benefits from successful approaches of phytoremediation
include healthier soil, promoting and sustaining indigenous microbial communities that are essential for long-term bioremediation
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Fig. 1. Schematic representation of uses of plant growth-promoting bacteria and AM fungi in bioremediation processes.
of the soil, and creation of a more pleasing landscape, compared
with ugly contaminated areas (Mendez and Maier, 2008). However,
often the plants needed for phytoremediation cannot establish in
degraded systems. Also, when established, they do not perform well
under adverse environmental conditions, such as excessive concentrations of contaminants, extreme high and low pH, deficient
supply of nutrients, poor or no soil structure, and severely damaged microbial communities. Under these conditions, inoculation
of PGPB and sometimes AM fungi assist in phytoremediation. PGPB
are experimentally employed to address environmental degradation, such as revegetation and reforestation programs in areas of
eroded soils, biological treatment of wastewater, phytoremediation and phytostabilization of abandoned areas, and restoration of
mangroves (Bashan et al., 1999; de-Bashan et al., 2002a; Duponnois
and Plenchette, 2003; Grandlic et al., 2008; Hernandez et al., 2006;
Kuiper et al., 2004; Lebeau et al., 2008; Ma et al., 2011; Stout and
Nüsslein, 2010; Sundararaman et al., 2007; Xue et al., 2009).
The aim of this review is to comprehensively describe the state
of the art in the use of PGPB in revegetation and reforestation of
areas undergoing desertification and contaminated sites, phytostabilization of mine tailings, phytoextraction of heavy metals and
organic contaminants, breakdown of contaminants using selected
plants, reforestation of mangroves, and enhancement of tertiary
wastewater treatment (Fig. 1). Additionally, we present research
venues likely to yield significant advances in knowledge to a level
of practical use of these emerging technologies.
2. Environmental applications of PGPB
2.1. PGPB for revegetation and reforestation of desertified lands
Soils in sub-humid, semi-arid, and arid areas are degraded by
human activities (Wang et al., 2004) in a process called desertification. Desertification is an increasing major problem that reduces
arable lands, increases soil erosion and flooding, reduces agricultural and forest production, reduces rural populations, and
increases respiratory health from dust pollution (McTainsh, 1986;
Ortega-Rubio et al., 1998; Wang et al., 2004). Natural revegetation
in deforested deserts is either very slow, minimal, or does not
occur. These are common worldwide problems, which are particularly severe in the Sahel of Africa and northeastern China. In
the Americas, desertification is prevalent in the southwestern USA,
northwestern Mexico, and arid regions of the Andes.
Desertified areas have hard-to-improve soil characteristics that
make restoration difficult. By default, restoration of severely
degraded deserts with shrubs, trees, and cacti is always difficult
and, in many cases, only marginally successful (Banerjee et al.,
2006; Bean et al., 2004; Glenn et al., 2001; Roundy et al., 2001)
due to several constrains. These constraints include: (1) the nurse
tree system governing natural vegetation is often destroyed and
the topsoil, with its beneficial microorganisms, are eroded by wind
and water, (2) organic matter is very low, soil structure is degraded,
and (3) nutrients and water are usually in short supply. While
these limitations can be partly overcome by application of compost, sludge from wastewater, and chemical fertilizers, these are
costly and impractical on a large scale. Consequently, natural succession and revegetation in these arid zones, if it occurs, is very
slow. Establishing vegetation can be difficult, takes considerable
time, and are labor intensive. Any attempt to restore native plants
should also consider restoring the beneficial microflora associated
with trees and shrubs and provide organic matter (Bacilio et al.,
2006; Grandlic et al., 2008; Velázquez-Rodríguez et al., 2001).
Various rehabilitation strategies have addressed restoration of
vegetation in desertified areas. These include reforestation with
native and exotic plants (Miyakawa, 1999; Moore and Russell,
1990), urban, agricultural, and pastoral coverage (Portnov and
Safriel, 2004), compost or sludge amendments to increase organic
matter and water-holding capacity (Mendez et al., 2007), and inoculation with plant growth-promoting microorganism (Grandlic
et al., 2008).
Reforestation of degraded lands undergoing erosion (Grover
and Musick, 1990) involves at least three stages (Fig. 2). First, it
is necessary to establish fast cover with native annuals to reduce
erosion, a task difficult to achieve when rainfall is very low or
unpredictable (Banerjee et al., 2006; Roundy et al., 2001). Second, a
cover with shrubs for short and medium term reforestation must be
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Fig. 2. Potential rehabilitation strategies to address desertification of eroded land.
established (Bean et al., 2004). Third, a long-term soil erosion prevention program, using long-lived and slow-growing trees, must be
established; this includes tree-shaped cacti, such as the giant cardon cactus (Pachycereus pringlei) (Bashan et al., 1999, 2000b), the
world’s most massive cactus, and several other large and long-lived
cacti and long-lived leguminous trees, such as mesquite (Drezner,
2006).
One of the fundamental theories about the recurrent failure of
natural revegetation in eroded desert areas and the difficulties to
establish planting or seeding native plants is that the topsoil has lost
its beneficial plant-associated microorganisms and, consequently,
part of its fertility and growth potential. Even during occasional
years of plentiful rainfall, when establishment of desert plants is
more likely (Drezner, 2006), water alone does not substitute for the
loss of soil fertility and microbial communities. At least some essential plant growth-promoting microorganisms should be artificially
reintroduced (Fig. 3).
PGPB and AM fungi are beneficial in harsh and limiting environments because of their role in alleviating stress in plants (Sylvia
and Williams, 1992). For example, PGPB of the genus Azospirillum exert their main growth-promoting effects when plants are
stressed in saline soils (Bacilio et al., 2004), by drought (Creus
et al., 1997, 1998), by excessive humic acids (Bacilio et al., 2003),
under extreme pH (de-Bashan et al., 2005), and presence of heavy
metals (Belimov and Dietz, 2000). Hyphae of AM fungi permeate large volumes of soil (Camel et al., 1991), interconnect root
systems of adjacent plants, which facilitate exchange of nutrients
between them, and contribute to plant growth and soil structure
as intimate associates with living cells within the roots and in
the soil (Bethlenfalvay and Schüepp, 1994; Bethlenfalvay et al.,
2007; Wright and Upadhyaya, 1998). AM fungi are recognized as
an essential component of plant–soil systems in deserts (Bashan
et al., 2000b, 2007; Bethlenfalvay et al., 1984; Carrillo-Garcia et al.,
1999; Cui and Nobel, 1992; Nobel, 1996; Requena et al., 2001).
PGPB do not yet have similar recognition and are used only at the
experimental level.
The use of PGPB for revegetation has several benefits: (1)
reduces costs of bioremediation by decreasing the amount of fertilizer and compost; (2) allows establishment of plants in eroded
zones where they had previously grown; and (3) improves plant
health and growth performance in eroded zones and enhances their
tolerance to drought and salinity.
Two strategies are used in the search for PGPB compatible with
eroded zones. One is to isolate native PGPB from the soil or from
plants already growing there, propagate the PGPB, and use the PGPB
as inoculants. This is a labor intensive procedure that has been successfully tried and explained below to remediate mine tailings. The
other approach examines isolates from the large number of available and proven PGPB strains, mainly used in agriculture and use
them as inoculants in revegetation efforts.
Several studies, described in more detail later, document the use
of the well-known PGPB Azospirillum brasilense of agricultural origin for revegetation of degraded desert lands that have lost their
capacity to support regeneration. These areas remain barren for
decades (Bacilio et al., 2006; Bashan et al., 1999; Carrillo-Garcia
et al., 2000). A. brasilense has proved to be an outstanding PGPB
in hundreds of agricultural studies and was recently commercialized to use as an inoculant in crop cultivation (Díaz-Zorita and
Fernández-Canigia, 2009). It possesses multiple growth-promoting
mechanisms: produces phytohormones (IAA, gibberellins, nitric
oxide), has significant diazotrophic capacity, and participates in
several small-magnitude mechanisms that work together or in tandem to enhance plant growth (Bashan and de-Bashan, 2010). It
plays a role in increasing acidification of inoculated rhizosphere
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Fig. 3. Contributions and interactions of plant growth-promoting microorganisms and organic amendments during revegetation and reforestation of eroded soils.
of grains and cardon cactus that solubilize essential nutrients for
plant growth that normally have extremely low bioavailability in
alkaline desert soils (Carrillo et al., 2002).
Initial studies demonstrated that cells of A. brasilense inoculated
on giant cardon, the world’s largest cactus that stabilize topsoil
in its usual desert scrub habitat, improve plant growth characteristics, are strain dependent, and are recoverable for at least 300
days following inoculation (Puente and Bashan, 1993). This PGPB
was demonstrated in a field trial in Mexico, where three species
of cacti had a significantly higher survival rate (76%), compared to
non-inoculated controls; <2% of the controls survived beyond 3.5
years. Plants in this trial were inoculated three times each year for 2
years; inoculation increased plant biomass and soil characteristics
in the experimental plots significantly improved. The most significant outcome from this trial was the great reduction in soil erosion
and reclamation of topsoil (Bashan et al., 1999).
Growth chamber tests showed that A. brasilense enhances
enzymes in the phosphogluconate pathway and growth of
mesquite seedlings (Prosopis articulata) that is cultivated in poor
soils (Leyva and Bashan, 2008). Soil quality may play a role in the
effectiveness of inoculated PGPB. A. brasilense had no effect on the
growth of the giant cardon cactus in rich soils found in desert
resource islands. At the same time, root and above-ground growth
of inoculated plants increased linearly after inoculation, enhancing
dry shoot mass by 60% and root length by 100% in the poorest quality soil (Carrillo-Garcia et al., 2000). Similarly, when A. brasilense
was used as an inoculant combined with compost in an attempt to
mimic resource island soil, the outcome from inoculation was small
compared to the effects of adding compost. Furthermore, PGPB
treatments were not significant at higher application rates of compost (Bacilio et al., 2006). These studies emphasize the importance
of assessing soil quality before PGPB inoculations are assessed.
Under screenhouse cultivation common to these trees in
nurseries, three slow-growing leguminous trees used in desert
reforestation and urban gardening in arid northwestern Mexico
and southwestern USA were measured to determine whether
growth was promoted by inoculation with PGPB (A. brasilense and
Bacillus pumilus), unidentified arbuscular mycorrhizal (AM) fungi
(mainly Glomus spp.), and a small supplement of common compost. Mesquite (P. articulata) and yellow palo verde (Parkinsonia
microphylla) had different, positive responses to several parameters, while blue palo verde (Parkinsonia florida) did not respond.
After cultivation for 10 months, survival of the latter two species
was >80% and mesquite was almost 100%. Inoculation with growthpromoting microorganisms induce significant effects on the gas
exchange of leaves of these trees, measured as transpiration and diffusion resistance, when these trees were cultivated without water
restrictions (Bashan et al., 2009b).
In the field, using the same combination of inoculants as in the
greenhouse trials, seven field trials were undertaken with cardon
cacti and three species of leguminous trees. The objective was to
avoid the lengthy process of succession in deserts, usually required
for establishing climax-stage plants such as cardon. This normally
require establishment of cardon seedlings growing under leguminous nurse shrubs and trees, such as mesquite. Mesquite prevents
soil erosion for several years or several decades, until cardon eventually replace them and will stabilize the soil for centuries (Bashan
et al., 1999, 2000b; Carrillo-Garcia et al., 1999; Solís-Domínguez
et al., 2011; Stutz et al., 2000; Titus et al., 2003).
In these field experiments, association of cardon with any nurse
shrub or tree increases survival and enhanced growth. For cardon
growing in isolation, compost, AM fungi, or combined treatments
increased survival. For cardon, no treatment influenced growth
during the first 3 months after transplanting. Later, all treatments,
including PGPB, except for AM fungi, enhanced growth. However,
2 years after transplanting to the field, enhanced growth by AM
fungi was significant. This field study proposed that young leguminous shrubs and trees enhance survival and growth of cardon,
depending on the combination of leguminous nurse plant and cactus. Additional treatments, such as compost or PGPB, can either
amplify or attenuate the effect (Bashan et al., 2009a). This study
also demonstrated that in a reforestation program, nurse plants
can be very young. In nature, associations of young nurse plants
with cardon are uncommon; only plants >20 years have nurslings
(Carrillo-Garcia et al., 1999).
This planting strategy (cardon-leguminous tree) may serve as a
shortcut for establishing cardon seedlings in the field. In the early
years, nurse plants are much larger than young cardon, shade them
and consequently make the water underneath their canopy more
available. Although additional treatments of compost and growthpromoting microorganisms are essential for survival and development of cardon growing without nurse trees, the effect of mesquite
on cardon development is more important over the long-term
than any microbial treatment provided when seedlings are planted.
Furthermore, over time, even plants without inoculation of AM
fungi become mycorrhizal because there are sufficient indigenous
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AM fungi (Bashan et al., 2000b). The additional treatments had a
synergistic effect only in the case of mesquite, the most common
nurse tree for cardon (Bashan et al., 2009b).
In other field experiments, survival of the three leguminous
trees (mesquite and two species of palo verde) was marginally
affected by supplements after 30 months; survival was in the range
of 60–90% of the trees, depending on the species, where all young
trees survived >3 months under cultivation. Mesquite and yellow
palo verde responded (height, number of branches, and diameter of
the main stem) positively to inoculation with PGPB, AM fungi, and
compost supplement, but blue palo verde did not respond to most
treatments. Response, in terms of specific plant growth parameters to specific inoculations or amendments, depended mainly on
the tree species. This field study demonstrated that restoration of
severely eroded desert lands is possible with native leguminous
trees supported with microbial agents and compost to increase
soil fertility and microbial activity near the trees (Bashan et al.,
unpublished).
Taken together, application of all amendments in the field,
as a single treatment, did not yield, as was initially assumed,
synergistic or cumulative effects. Rather, the usefulness of each
amendment was mainly related to particular species, not as a
universal combination of amendments. Similarly to the desert
restoration, Quoreshi et al. (2008) demonstrates selective effects of
a microbial amendment when conifers and hardwoods in Canada
were inoculated with several mycorrhizae fungi at the nursery.
Siviero et al. (2008) shows similar selective effects on the tropical leguminous tree Schizolobium amazonicum, commonly used in
agroforestry in the Amazon region, when inoculated with three
arbuscular mycorrhizal fungi (Glomus clarum, Glomus intraradices,
and Glomus etunicatum) associated with three strains of nitrogenfixing bacteria, two Rhizobium spp. and one Burkholderia sp.
Another example from a desertified, semi-arid area of southeastern Spain demonstrated that several exotic and native woody
legumes could be used in revegetation programs. Screening for the
appropriate combination of plant and microsymbiont (mycorrhizae
and rhizobia) was previously tested. The results of a 4-year field trial
showed that only the native shrubby legumes were able to become
established under local environmental conditions; hence, a specific
reclamation strategy was recommended, which included inoculation with selected rhizobia and AM fungi for seedlings intended for
revegetation (Herrera et al., 1993). In a program of reforestation
with Pinus halepensis in degraded gypsiferous soil, two techniques
commonly used in restoring soil were compared: (1) inoculation
with selected strains of PGPB and (2) creation of a soil amendment
from urban waste. One year after planting, inoculation with two
strains of PGPB that had previously been used to enhance growth
of Pinus spp. did not affect pine growth or the nutrient content of
needles. However, significant increases in growth occurred under
the soil amendment treatment. Rincon et al. (2006) concluded that
scaling up greenhouse techniques, commonly used for PGPB of
leguminous trees, does not necessarily guarantee that other tree
species will receive the same benefits. In Korea, stimulation of
growth of wild plants using several species of PGPB was tested on
a barren area; average shoot and root lengths and total dry weight
of the plants grown in inoculated soil was far greater than plants
grown in uninoculated soil (Ahn et al., 2007).
Other studies illustrate the value of using native PGPB. PGPB
that were obtained in an arid region improved rooting of mesquite
cuttings (Felker et al., 2005). Cacti growing as pioneer plants on
rock and cliffs devoid of soil materials provided strains of PGPB
isolated from the surface of cardon roots (Puente et al., 2004a),
endophytes of cardon (Puente et al., 2009a), and the surface of a
small cactus, Mammillaria fraileana (Lopez et al., 2011). These bacteria weather the rock where the cacti grow, which release essential
minerals and produced small amounts of soil material that is later
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washed down slopes and accumulate at lower elevations to support desert plants that are not capable of pioneer growth on rock
(Lopez et al., 2009). When endophytic bacteria are eliminated from
the cacti seeds, seedlings grew more slowly. When sterilized seeds
were inoculated with these bacteria and cultivated in pulverized
rock for 1 year, almost all of these bacteria enhanced plant growth
in these harsh environments (Puente et al., 2004b, 2009b).
2.2. Revegetation with AM fungi and PGPB
In revegetation projects, using AM fungi alone or with PGPB is
common in commercial reforestation of shrubs and trees in temperate areas (Chanway, 1997; Duponnois and Plenchette, 2003;
Shishido et al., 1996), but far less in reforestation of deserts. Colonization by AM fungi of desert plants is found in numerous common
trees and cacti (Bashan et al., 2000b; Carrillo-Garcia et al., 1999)
and relic trees (Bashan et al., 2007). Application of AM fungi is still
uncommon, although reforestation is now a priority in all Sahelian
and Sudano-Sahelian countries, northern Mexico, and Australia. It
is likely that AM fungi play a similar role in desert plants as in agricultural crops growing in arid areas because plants rely on these
symbiotic fungi to absorb nutrients, especially phosphorus and
mineral transport in general. Yet, scientific data is in short supply.
Many times, reforestation is done with fast-growing Acacia trees.
The ability of Acacia to grow in low-nitrogen soils depends on their
ability to form root symbiosis with both rhizobia and interaction
with AM fungi. Much of the nitrogen provided in the symbiosis is
returned to the soil through leaf fall; in turn, the humus improves
fertility and physical properties.
There are only a handful of examples of these multiple inoculations for restoring vegetation. (1) In semi-arid areas of southern
Europe, a combination of AM fungi, rhizobia, and PGPB was used to
enhance growth of the woody shrub Anthyllis cytisoides using native
(Glomus coronatum) and exotic (G. intraradices) AM fungi. In revegetation programs, this shrub had greater root and shoot dry weight
and enhanced root nodulation. PGPB also enhanced seedling germination of this shrub. Native AM fungi were more effective than
non-native fungi species, indicating that native isolates are more
suitable to specific ecosystem conditions (Requena et al., 1997). (2)
In a desertified ecosystem, specific combinations of AM fungi, PGPB,
and Rhizobium spp. enhanced plant growth and uptake of nutrients. During a 5-year study, inoculation with AM fungi and rhizobia
enhanced establishment of key plant species, as well as increased
soil fertility (Requena et al., 2001). (3) In southern Senegal, ectomycorrhizal symbiosis of the strap wattle Acacia holosericea, a shrub
from Australia, was directly promoted by 14 bacterial strains isolated from the soil mycorrhizosphere and the roots. Shoot mass was
enhanced by even more strains, all indicating the potential of some
bacteria to help mycorrhiza in reforestation programs in the tropics (Founoune et al., 2002). (4) The bacterium Pseudomonas monteilii
promoted mycorrhiza colonization of A. holosericea of all species of
Acacia (Duponnois and Plenchette, 2003). (5) In a semi-arid area in
Southern Europe, a genetically modified bacterium performed less
well to help mycorrhiza than wild strains (Valdenegro et al., 2001).
(6) In tropical Southeast Asia, forest product companies used plant
fast-growing and non-indigenous tree species to reduce regional
shortages of wood by cultivating black wattle or mangium, Acacia
mangium, which grows rapidly on infertile soils and produces abundant wood in short rotations. Symbiosis between A. mangium and
rhizobia and AM fungi is a management strategy where soils limit
plant growth. Black wattle seedlings are sometimes inoculated with
rhizobia and AM fungi in the nursery during early stages of growth.
Aeroponics was used to produce saplings associated with AM fungi
that were successfully transferred to the field (Martin-Laurent et al.,
1999a,b).
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In conclusion, there is no doubt that associative PGPB, rhizobia, and AM fungi can be successfully used in revegetation and
reforestation programs. Applications should consider inherent limitations of PGPB, which work best on seedlings during initial stages
of growth, and using rhizobia and AM fungi that establish longlasting associations with trees and shrubs in these programs. So
far, no general interaction between a PGPB and a number of plant
species was established on a long-term basis in the field, although
some beneficial associations were demonstrated under controlled
conditions. Although there are experimental indications that inoculation of a consortium of species might function better, it is not
the norm. Each combination of plant species, PGPB, and AM fungi
should be tested separately before attempting large-scale revegetation programs. This fact limits quick application of this promising
technology.
Mainly because of scarcity of studies on this topic, there are
numerous open questions; some have answers only in agricultural
studies. Questions include: (1) are mycorrhizae-helping bacteria
useful in revegetation of eroded lands? (2) How do the PGPB
enhance AM fungi infection in plants in the environment? (3) Are
the responses of AM fungi to PGPB similar to those of ectomyccorrhiza to PGPB? (4) Because triple partnerships (plant–PGPB–AM
fungi) and quadruple partnerships (when rhizobia are used in
legumes) produce a multiple of unpredicted interactions and possibly complex mechanisms of action, would it be better to use only
one type of microorganism? (5) Is it necessary to inoculate AM fungi
when the soil has enough AM inoculum-potential, allowing inoculation with only PGPB to suffice? (6) Because inoculation of bacterial
consortia has not proven to be optimal in some cases, why does the
environmental restoration industry use consortia as recommended
inoculants?
2.3. PGPB for restoration and reforestation mangrove ecosystems
Mangrove forests are vital tropical marine coastal ecosystems
found at the intertidal zones of estuaries, backwaters, deltas, creeks,
lagoons, marshes, and mud flats in tropical and subtropical regions
(Spalding et al., 2010). Worldwide, mangroves are continually cut
for coastal urban development and aquaculture facilities; since
the 1980s, over 25% of mangroves have been removed (Adeel and
Pomeroy, 2002; FAO, 2003; Rönnbäck, 1999). Mangroves provide
an essential environmental service, acting as safe havens and breeding grounds for countless marine species and waterfowl and are
an irreplaceable prerequisite for coastal fisheries in the tropics
(Holguin et al., 2001). Mangroves create a critical barrier that protects coastal settlements and rice fields from tropical storms and
tsunamis, mainly in Eastern Asia. Although mangroves in the wet
tropics are easy to reforest (Primavera et al., 2004), mangroves in
arid and semi-arid regions are especially sensitive and have difficulty regenerating after disruption (Toledo et al., 2001; Vovides
et al., 2011a). The highly productive and diverse microbial communities living in mangrove ecosystems continuously transform
nutrients from dead mangrove vegetation into sources of nitrogen, phosphorus, and other mineral nutrients that are used by
the plants. In turn, exudates of plant roots serve as a food source
for the microbes and organisms higher up the food chain (Bano
et al., 1997; Kathiresan and Bingham, 2001; Sahoo and Dhal, 2009).
Holguin et al. (2001) predicted that, without restoring the microbial food web, restoration of mangroves would be very difficult,
if not impossible. Efforts are currently underway to characterize
these ecosystems, including their microbial communities and the
microbial processes, with goals of developing successful restoration strategies. A secondary goal is to find microbial inoculants
that can be used in agriculture in saline soils (Holguin et al., 2006;
Sundararaman et al., 2007). These microbial communities include
nitrogen-fixing bacteria and nitrogen cycle process (Flores-Mireles
et al., 2007; Hoffmann, 1999; Holguin et al., 1992; Sengupta and
Chaudhuri, 1990; Toledo et al., 1995a; Vovides et al., 2011a,b),
phosphate-solubilizing bacteria (Rojas et al., 2001; Vazquez et al.,
2000), methane generating bacteria (Giani et al., 1996; Mobanraju
et al., 1997; Strangmann et al., 2008), sulfate-reducing bacteria
(Lokabharatbi et al., 1991; Sherman et al., 1998), and general heterotrophic populations (Bano et al., 1997; Gonzalez-Acosta et al.,
2006).
Bashan and Holguin (2002) initially proposed the use of PGPB
for reforestation of impaired mangroves and outlined the possible microbial interactions in this process. They identified a
collection of isolates that possessed beneficial growth-promoting
traits, including nitrogen fixation, phosphate solubilization, and
phytohormone production (Fig. 4). Work applying PGPB to mangrove plants revealed that inoculation with the diazotrophic
cyanobacterium Microcoleus chthonoplastes, isolated from aerial
roots (pneumatophores) of mangroves, increased root colonization
of this bacterium on black mangroves and nitrogen accumulation
in planta (Bashan et al., 1998; Toledo et al., 1995b). This cyanobacterium and several other PGPB from mangroves have a beneficial
side effect when introduced to dwarf saltwort Salicornia bigelovii,
a halophytic annual that inhabits coastal marshes as well as mangroves. Inoculation enhanced plant biomass by 70% and improves
the quality of the essential oils in this saltwort (Bashan et al., 2000a;
Rueda-Puente et al., 2003).
Introducing the agricultural PGPB A. brasilense to mangrove
seedlings demonstrated that two species, A. brasilense and Azospirillum halopraeferens, have the ability to colonize root surfaces
of black mangrove seedlings. A. halopraeferens was a better colonizer and its presence on root surfaces was confirmed by
scanning electron microscopy (Puente et al., 1999). Additional
laboratory and greenhouse studies use screening methods that
isolate and characterize large collections of potential PGPB (diazotrophs, denitrifying bacteria, phosphate-solubilizing bacteria)
from mangroves and the conditions that control proliferation and
interaction among different species of bacteria (Flores-Mireles
et al., 2007; Holguin and Bashan, 1996; Holguin et al., 2001;
Rojas et al., 2001; Vazquez et al., 2000). For example, Kathiresan
and Masilamani Selvam (2006) screened 48 isolates from mangroves and reported that two isolates demonstrated potential to
increase plant growth by >100%; hence, they are excellent candidates for reforestation projects. El-Tarabily and Youssef (2010)
studied an efficient phosphate-solubilizing bacterium, Oceanobacillus picturae, isolated from the rhizosphere of the gray mangrove
Avicennia marina. In the greenhouse, inoculation of O. picturae
into sediments amended with rock phosphate led to growth of
roots and shoots of seedlings that were significantly better than
seedlings in sediment amended only with rock phosphate. This bacterium significantly increased available phosphate in the sediment,
decreased the pH of the sediment, positively enhanced nutrient
uptake parameters in roots and shoots, enhanced morphological
parameters of the plants, and increased photosynthesis, compared
to plants grown in the identical sediment but without the bacterium. Similarly, several Azotobacter species (A. chroococcum, A.
virelandii, and A. beijerinckii) inoculated on red mangrove seedlings
(Rhizophora) significantly increased average root biomass, root
length, leaf area, shoot biomass, and concentrations of chlorophylls and carotenoids, compared to controls (Ravikumar et al.,
2004). Inoculation of black mangroves with undefined phosphate
solubilizers and nitrogen fixers improved growth of mangrove
seedlings in nurseries (Galindo et al., 2006).
In summary, recent work with PGPB shows that mangrove
rhizosphere bacteria can be used to enhance reforestation with
mangrove seedlings. This can be done by inoculating seedlings
with PGPB that participate in one or more of the microbial cycles
of the ecosystem. Still, the current state of knowledge on the
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Fig. 4. Documented and proposed groups of bacteria and pathways that occur in mangrove ecosystems and have potential as a source for plant growth-promoting bacteria
in mangroves and microbial sustenance of the mangrove ecosystem. This is a revision of the model originally proposed by Holguin et al. (2001) and Bashan and Holguin
(2002).
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microbiology of mangrove ecosystems leaves some fundamental
questions unanswered. (1) Does nitrogen fixation and phosphate
solubilization in the ecosystem significantly contribute to the vitality of the trees? (2) Do mangrove specific PGPB exist or are
these strains common marine bacteria with PGPB traits? (3) Is
it possible to enhance mangrove plant growth with terrestrial,
agriculture-originated salt-tolerant PGPB? (4) Should restoration of conditions for microbial activity precede reforestation or
are these processes interlinked? (5) Are techniques for inoculation with PGPB of terrestrial plants also applicable to wetland
plants?
2.4. PGPB for enhancing phytoremediation of contaminated soils
Phytoremediation is an in situ, solar-powered comprehensive
strategy for bioremediation of environmental problems by using
plants and their associated microorganisms to extract, sequester, or
detoxify pollutants. The plants mitigate the problems; environmentally invasive excavation of the contaminated material to dispose of
it elsewhere is avoided. Plants are able to contain, degrade, or eliminate metals, pesticides, explosives, solvent, crude oil, and many
industrial contaminants. Phytoremediation requires minimal disturbance and maintenance of a site and is a clean, cost-effective,
environmentally friendly technology, especially for treatment of
large contaminated areas with diffuse pollution. Many successful
examples of phytoremediation are documented and employed in
the environmental cleaning industry (Macek et al., 2000; Suresh
and Ravishankar, 2004).
The major disadvantage of phytoremediation is that phytoremediation requires a long-term economic commitment and patience
by project managers for results because the process is dependent
on plant growth, tolerance to contaminants, and bioaccumulation capacity, all of which are slow processes. Phytoremediation
involving a combination of several independent approaches might
show an effect. These approaches include: (1) phytotransformation, which reduces toxicity, inactivates, or degrades contaminants
resulting from plant metabolism; (2) rhizodegradation, which
enhances soil microbial activity to degrade contaminants by rhizosphere bacteria; (3) phytoextraction, which absorbs contaminants
from the polluted soils and stores the substances in the plant
biomass, with a potential to recover and re-use valuable metals,
and (4) phytostabilization, which reduces mobility of toxic substances in the soils, as in the case of mine tailings. All these benefits
notwithstanding, on their own, plants have the disadvantage of
being inefficient. Even plants that are relatively tolerant of environmental contaminants often remain small in the presence of
contaminants and remove only small quantities per plant. To obtain
more efficient degradation of organic compounds, plants depend on
their associated microorganisms (Pilon-Smits and Freeman, 2006).
To improve this process, inoculation with PGPB that enhance plant
growth, especially under stressed conditions, offers advantages.
Inoculation increases plant biomass, making phytoremediation a
faster and efficient process and more attractive to the public (Glick,
2003).
2.5. Phytodegradation, phytotransformation, bioaugmentation,
rhizodegradation, and phytoextraction
Many organic pollutants are converted to harmless material
by the metabolism of plants and the rhizosphere microorganisms
living in association with the plant roots. Phytodegradation combined with bioaugmentation, microorganisms assisting the plants,
(Lebeau et al., 2008) is defined as rhizodegradation. Root exudates provide a nutrient-rich environment where microbial activity
is stimulated, which leads to more efficient degradation of pollutants. The root system also supports three-dimensional spread
of bacteria deeper into soils, penetrating otherwise impermeable
soil layers (Kuiper et al., 2001, 2004). Because some complex and
recalcitrant compounds cannot be broken down to basic molecules
(water, CO2 , volatile compounds) by plants or microbial enzymes,
phytotransformation represents a change in chemical structure
to a less harmful compound without complete breakdown of the
compound. In the final stage of phytotransformation, immobilization of the xenobiotic substance occurs within the plant, hence,
phytoextraction. Phytoextraction also occurs by direct uptake of
the contaminant if the metabolism of the plant is resistant to
its toxicity. Xenobiotic substances are polymerized within the
plant and develop a complex structure that is sequestered in
the plant and does not interfere with normal functioning of the
plant (McCutcheon and Schnoor, 2003; Mukhopadhyay and Maiti,
2010). In many cases, the effective solution combines several
of these processes. Because these techniques can last years or
decades, progress on many sites often includes combinations of
several techniques; therefore, it is difficult to estimate efficacy
of bioremediation by each approach on large contaminated sites.
Most published information deals with controlled and greenhouse
experiments.
2.6. PGPB for enhancing phytodegradation by increasing
rhizoremediation
The rhizosphere contains microbial populations that are several orders of magnitude greater than the surrounding bulk soil;
it is composed of numerous species capable of degrading xenobiotic substances (Donnelly et al., 1994; Macková et al., 2007;
Mendez and Maier, 2008). Along with higher capacity to degrade
substances, the rhizosphere of plants growing in contaminated
soils may serve as a reservoir of PGPB. Because this is a relatively recent concept, there is little information in the scientific
literature. Related information, not scientifically peer-reviewed, is
available on commercial websites. Kuiper et al. (2001) describes
efficient root-colonizing, pollutant-degrading bacteria inoculated
on a suitable plant for phytoremediation, these bacteria settle on
the roots with the indigenous bacterial population and enhanced
bioremediation. A combination of a plant and microbe was selected
that led to efficient degradation of naphthalene and protected
grass seed against high concentrations of naphthalene. In another
soil, purposely contaminated with creosote, inoculation of tall fescue (Festuca arundinacea) with bacteria that degrades polycyclic
aromatic hydrocarbons (PAH) and PGPB (Pseudomonas putida, A.
brasilense, and Enterobacter cloacae), the rate of removal of PAH
was substantially increased. Large-sized PAH were removed in the
presence of these PGPB because these specific bacterial species
mitigated stress in plants in general by ACC-deaminase activity,
a common mechanism in PGPB (Huang et al., 2004). In another
study, the PGPB, Pseudomonas spp., increased growth of the oil
plant canola and common reed Phragmites australis in the presence of copper or PAH (Reed and Glick, 2005; Reed et al., 2005). In
still another study, PGPB helped to degrade 2-chlorobenzoic acid
and oil-contaminated soils for growing the broad bean Vicia faba
and several forage grasses, but no clear relationship between added
disappearance of the pollutants and increased plant biomass was
found (Radwan et al., 2005; Siciliano and Germida, 1997). Bioremediation potential of the legume, goats rue Galega orientalis and
its symbiont Rhizobium galegae was assessed in soils contaminated
with benzene, toluene, and/or xylene (BTX). Good growth, nodulation, nitrogen fixation, and a strong rhizosphere occurred in soils
contaminated with oil or spiked with m-toluate, a model compound
representing BTX (Suominen et al., 2000).
Similar to rhizosphere PGPB, many endophytic PGPB assist
host plants to overcome contaminant-induced stress, which provided improved plant growth. In comparison with rhizosphere and
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Fig. 5. Restrictions to large-scale applications of phytoremediation of organic pollutants and possible solutions.
phyllosphere PGPB, endophytic PGPB are likely to interact more
intimately with their host. During phytoremediation of organic
contaminants in soils, plants further benefit from their endophytes,
which possess degradation pathways and metabolic capabilities
not inherent in the plant. This strategy leads to more efficient degradation and reduction of phytotoxicity and evapotranspiration of
volatile contaminants (Weyens et al., 2009). For example, tall fescue F. arundinacea grass can selectively enhance the prevalence
of endophytes containing pollutant catabolic genes in environments contaminated with different pollutants (hydrocarbons and
nitro-aromatics). Enrichment of endophytic catabolic genotypes is
plant- and contaminant-dependent (Siciliano et al., 2001). Barac
et al. (2009) found that, when remediation reduced BTX below
detectable levels, the capacity of the endophytic community in
poplars to degrade BTX disappeared. When the pea plant Pisum
sativum was inoculated with an endophyte originated from poplar
and capable of degrading the herbicide 2,4-D, it increased removal
of 2,4-D from the soil (Germaine et al., 2006).
There are several restrictions for large-scale application of phytoremediation of organic pollutants: (1) limits to the concentration
of contaminants that can be tolerated by plant species; (2) frequently, contaminants are not bio-available; and (3) dispersal of
the contaminant to the atmosphere (volatization) is not always
acceptable. The use of task-specific, genetically engineered plants
and modifying the rhizosphere for accelerated rhizodegradation
of persistent organic contaminants may solve these difficulties
(Doty, 2008; Dzantor, 2007). If no naturally occurring endophytes
are available that have the desired metabolic properties, general
endophytic bacteria can be isolated from the plants. The needed
degradation pathways can be added to these endophytes by genetic
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manipulation and these modified endophytes can be inoculated
by vacuum infiltration (Puente and Bashan, 1993) into the seeds
of the host plants to serve as PGPB. Engineered endophytes will
improve phytoremediation by complementing the metabolic properties of their host plant. Evidence for this approach was provided
by inoculating yellow lupine Lupinus luteus (Barac et al., 2004) and
poplar Populus sp. (Taghavi et al., 2005) with endophytic bacteria able to degrade toluene. This reduced toluene phytotoxicity
and significantly lowered evaporation of toluene. In another study,
a plasmid was transferred from Burkholderia cepacia to an endophytic PGPB strain of this species and this protected the new host
against toluene toxicity and reduced evaporation of toluene (Glick,
2004). Newman and Reynolds (2005) engineered endophytes that
increased plant tolerance to toluene and decreased transpiration of
toluene to the atmosphere.
In summary, rhizosphere and endophytic PGPB have potential
to enhance biodegradation of organic pollutants. The advantages of
endophytic PGPB over rhizosphere PGPB give additional perspective in the use of the former as the method of choice for further
development.
2.7. Phytoextraction
Phytoextraction (also known as phytoaccumulation, phytoabsorption, and phytosequestration) is a green in situ technology
that uses plants to remove contaminants from the environment by
concentrating them within the biomass of the plant. The process
has been studied mostly for extraction of heavy metals from soils
and water containing moderate concentrations of contaminants.
The purpose for this technology is to reduce concentrations of metals in contaminated soils to a level that permits the soil to be used
without danger in agriculture, horticulture, forestry, grazing, or
urban uses. Within the plant biomass, the contaminant is more concentrated than in the environment, which facilitates safe disposal.
Phytoextraction is best performed by growing plants that are highly
metal-accumulating, the so-called hyper-accumulators. These are
plants that can tolerate and absorb higher amounts of toxic metals than most plants. A conditioning fluid containing a chelator,
which enhances mobilization of metals may be applied to a contaminated site to mobilize the metals and help the plants absorb
the metals more easily. So far, large transgenic plants have shown
no advantage in phytoextraction, compared to the high potential
of genetically unmodified plant species (Cunningham et al., 1995;
Ernst, 2005; Pilon-Smits, 2005). The limitations of phytoextraction,
such as bioavailability, uptake, and toxicity are well recognized and
require significant optimization steps to make it a widely useful
technology (Lebeau et al., 2008).
Phytoextraction is usually a long-term process that cannot
entirely eliminate the contaminant (Fig. 5). Extraction has been
demonstrated only for some metals. Although numerous plants can
serve as metal absorbers to some extent, many of the 400 known
hyper-accumulating plant species belong to the Brassicaceae family with some plants in an additional 10 families (Gratão et al.,
2005; Nanda Kumar et al., 1995; Prasad and Freitas, 2003). Genetic
manipulation of plants and its associated microbial communities
(rhizosphere engineering) has been proposed to optimize phytoextraction. These include: isolation of competent plant-associated
rhizosphere bacteria and loading them with metabolic pathways
that allow the synthesis of natural chelators to improve metal
bioavailability or sequestration systems to reduce phytotoxicity.
This is followed by inoculating the genetically changed bacteria to plants growing on contaminated soils (Lodewyckx et al.,
2001; Valls and de Lorenzo, 2002). Endophytes possessing a metalresistance or sequestration system can lower metal phytotoxicity
of the plants and transfer metals to aerial parts (Weyens et al.,
2009).
Some examples demonstrate this principle: uptake of cadmium
and zinc by the hyper-accumulating perennial herb Sedum alfredii
can be increased after plants are inoculated with endophytic bacteria equipped with the appropriate metal-resistance/sequestration
system (Li et al., 2007). When yellow lupin L. luteus was grown
on a nickel-enriched substrate and inoculated with the engineered
nickel-resistant endophytic bacterium B. cepacia, there was a 30%
increase of nickel in the roots, whereas nickel in the aerial parts
remained comparable with control plants (Lodewyckx et al., 2001).
Since plants absorb contaminants from soil or water through roots,
biological factors that modify root metabolism and enlarge the root
system, such as inoculation with PGPB, can enhance phytoextraction (Ma et al., 2011; Stout and Nüsslein, 2010). AM fungi have been
suggested for improving phytoextraction (Cornejo et al., 2008; Fa
et al., 2007), but this topic is outside the scope of this review.
Depending on the economic value of the metal, accumulated
toxic metals in hyper-accumulators can be harvested for metal
recovery and the leftover biomass composted. On the less favorable
side of phytoextraction, apart from inherent capacity to accumulate
contaminants, many hyper-accumulators produce a small biomass
and have limited ability to extract more than one or two metals. They are also sensitive to other metals that are present in
abundance. In heavily contaminated soils, growth of these plants
can be retarded or have minimal uptake so that phytoextraction
projects become impractical. Inoculation with phytostimulating
PGPB might be a solution in these cases.
PGPB and AM fungi improve rates of phytoextraction in two
main ways: (1) enhance general growth of plant. With a larger
biomass, more contaminants can be absorbed; and (2) increase
metal mobilization by microbial metabolites (Lebeau et al., 2008).
The ideal PGPB might do both (Rajkumar and Freitas, 2008a,b;
Saravanan et al., 2007; Sheng and Xia, 2006), but usually one
mechanism in each microorganism was studied. There are many
examples demonstrating these abilities. Inoculation with rhizobia
enhanced dry plant biomass of soybean in arsenic-contaminated
soils (Reichman, 2007). Root elongation of ferns and arsenic accumulation increased after inoculation with rhizospheric bacteria
(Jankong et al., 2007). Aerobic bacteria Variovorax spp. enhanced
root elongation of Indian mustard (Brassica juncea) in the presence
of cadmium (Belimov et al., 2005). Following inoculation with bacterial strains, large increases in several metals were sequestered
without enhancing biomass (Brunetti et al., 2011; Hoflich and
Metz, 1997; Rajkumar and Freitas, 2008a,b; Reed and Glick, 2005;
Whiting et al., 2001). Inoculation might improve overall plant nutrition, as is common of PGPB. Several rhizosphere strains enhanced
volatilization and accumulation of selenium and mercury in plant
tissues in an artificially constructed wetland (de Souza et al.,
1999a,b). Under hydroponic conditions, simultaneous addition of
EDTA and indole-3-acetic acid (IAA) synthesized by rhizobacteria increased extraction of lead by a factor of 28, in contrast
to only a factor of 6 with EDTA (Lopez et al., 2005). Several
isolates of rhizosphere bacteria with common plant growthpromoting traits enhanced extraction of cadmium and zinc by
willow seedlings using a variety of mechanisms, but not by producing IAA and 1-amino-cyclopropane-1-carboxylic acid deaminase
(ACC deaminase), which are common mechanisms in agricultural
PGPB (Kuffner et al., 2008). A surprising finding was that in vitro
experiments of mobilization of metals predicted the effects of
bacteria on willows more reliably than standard tests for plant
growth-promoting activities (Kuffner et al., 2010). A similar case
involved three rhizosphere bacteria isolated from the root of the
hyper-accumulator yellowtuft Alyssum murale, an annual that significantly increased uptake of nickel into the aerial parts, while
untreated plants had no positive effect on extracting nickel from
soil (Abou-Shanab et al., 2003). When the hyper-accumulator S.
alfredii was inoculated with B. cepacia, growth, uptake of metal,
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greater translocation of metals from root to shoot, and stimulation of organic acid secretions occurred. There appears to be a
functional resistance to metals that chelate metal ions extracellularly, which reduces uptake and subsequent deleterious effect
on physiological processes in the root (Li et al., 2007). Increases in
solubilization of metals and subsequent plant uptake of these contaminants may be the result of organic acid production by some
PGPB (Saravanan et al., 2007). Many PGPB that are phosphate solubilizers produce organic acids (Lopez et al., 2011; Puente et al.,
2004a,b; Rodriguez et al., 2004, 2006; Vazquez et al., 2000). Several PGPB (Pseudomonas aeruginosa, Pseudomonas fluorescens, or
Ralstonia metallidurans) that are able to produce siderophores were
introduced to a maize field containing chromium and lead. R. metallidurans enhanced accumulation of chromium in the maize shoots
by a factor of almost five; similarly, P. aeruginosa increased uptake
of chromium into maize shoots at the same rates, but not in their
roots. Consequently, total uptake of metal by the whole plant usually decreases after inoculation with these bacteria (Braud et al.,
2009).
PGPB can provide protection to plants against metal toxicity.
For example, Kluyvera ascorbata, which contains the enzyme ACC
deaminase, protects Indian mustard B. juncea and rapeseed Brassica
campestris against nickel, lead, and zinc toxicity (Burd et al., 1998),
probably by reducing the stress caused by high ethylene content.
Root elongation of rapeseed Brassica napus is also stimulated by the
IAA that is synthesized by PGPB in soil contaminated with cadmium
(Sheng and Xia, 2006), as was an unidentified rhizobacterium on
Indian mustard roots (Belimov et al., 2005).
The best-studied PGPB are Gram-negative bacteria, but some
Gram-positive PGPB are also known for excellent biocontrol, plant
growth promotion, and bioremediation activity (Francis et al.,
2010). For example, at sites contaminated with nickel, inoculation
of Indian mustard with B. subtilis yielded high concentrations of
nickel in the plant tissues and increased plant biomass. This results
from a combined effect of bacterial hormone production, solubilization of inorganic phosphate, and adsorption of nickel (Khan
et al., 2009). Inoculation of Indian mustard with a nickel-tolerant
B. subtilis significantly enhanced plant growth and produced a protective effect against phytotoxicity from nickel, probably through
involvement of IAA in uptake of the metal (Zaidi et al., 2006). B.
pumilus and Rhodococcus spp. increased plant biomass, but not the
amount of metals accumulated by plants (Belimov et al., 2001,
2005; Safronova et al., 2006).
Some PGPB can degrade pollutants or prevent their accumulation in plant tissues. In soil contaminated with heavy metals,
Arthrobacter mysorens, which is resistant to cadmium and lead, can
improve plant growth in the rhizosphere of barley and prevent
accumulation of heavy metal in the plant tissues. Several Bacillus strains isolated from chromium-contaminated soil reduced the
highly toxic, mutagenic, and carcinogenic Cr6+ to the less toxic Cr3+
(Khan et al., 2009). Psychotrophic Rhodococcus erythropolis that was
isolated from a metal-contaminated site in the Himalayas had plant
growth-promoting traits and an ability to reduce chromate at low
temperatures of ∼10 ◦ C (Trivedi et al., 2007).
Excretion of root exudates can stimulate growth of specific, pollutant-degrading bacteria in the rhizosphere by secreting
phospholipid surfactants that make organic pollutants more bioavailable or by releasing secondary metabolites that induce
expression of genes with a capacity to degrade organic pollutants
(Pilon-Smits, 2005). For example: Rhodococcus spp. were the
most common group in the rhizosphere of trees that naturally
colonized and improved a PCB-contaminated site in the Czech
Republic (van der Geize and Dijkhuizen, 2004). Barley growing
in PAH-contaminated soils supported growth of a Mycobacterium
species capable of mineralizing the PAH (Child et al., 2007). Soils
contaminated with petroleum derivatives typically contain high
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concentrations of m-toluate. Although Pseudomonas spp. are the
best m-toluate degraders in the rhizosphere of oriental goat’s rue
G. orientalis grown on these soils, most isolates were Gram-positive
strains of the genera Rhodococcus, Arthrobacter, Bacillus, and Nocardia (Jussila et al., 2006).
In summary, phytoextraction of heavy metals and a few other
contaminants show promise, but not much capacity on a larger
scale than the laboratory and greenhouse. To optimize phytoextraction, a contaminated site should be considered as a large-scale
bioreactor supported by an approach where the microbial population of the rhizosphere is genetically engineered. Large-scale
development of phytoextraction will depend on: (1) reliability
and repeatability of the process, which yet needs to be demonstrated; (2) ease of application needs to be similar to that for
rhizobia and PGPB applied in agriculture; (3) capacity to clean up
poly-contaminated soils; and (4) economic value demonstrated by
comparing microorganism-assisted plants or plants not assisted
by microorganisms, as well as whether PGPB promote or do not
promote plant growth under adverse conditions.
2.8. Phytostabilization
Phytostabilization (also known as in-place inactivation or phytoimmobilization) is a long-term, unobtrusively green, in situ
approach to contain contaminants. When a soil is highly
contaminated with more than one metal (polymetallic soils), phytoextraction (described above) will last decades or more; thus,
this period is too long for an economically feasible technology
(Ernst, 2005). Phytostabilization can occur through metallic sorption, precipitation, complexing, or reduction of toxicity. The basis
for phytostabilization is that metals do not degrade, so capturing
them in situ is sometimes the best alternative at sites with low
contamination levels or vast contaminated areas where large-scale
removal or other in situ remediation is uneconomical. There is consensus that it is better to restrict a contaminant in one area than
to allow it to disperse to uncontaminated areas. A vegetative cap
that is sufficiently dense to prevent wind erosion and roots that
reduce water erosion can prevent dispersal to nearby urban and
agricultural areas and into aquifers. Plants may restrict pollutants
by creating a zone around the roots where the pollutant is precipitated and stabilized. Phytostabilization does not absorb pollutants
into plant tissue, but sequesters pollutants in soil near the roots,
becoming less bio-available and reducing exposure to humans, livestock, and wildlife. The most common application is containment of
mine tailings (McCutcheon and Schnoor, 2003; Mench et al., 2006;
Mendez and Maier, 2008; Mukhopadhyay and Maiti, 2010; Petrisor
et al., 2004; Xue et al., 2009; Zhuang et al., 2007).
2.8.1. PGPB in programs for phytostabilization of mine tailings
Mainly in deserts, wind-blown toxic dust and pollution of
ground water from large mounds of tailings from abandoned and
producing mines are potentially a long-term health hazard to
nearby urban populations. Tailings, lacking plant cover and soil
structure, are easily transported by wind and rain; thus, tailings
serve as a continuous source of metallic pollution (Pilon-Smits,
2005). Phytostabilization is an economical strategy for using native
plants as ground cover to prevent erosion and reduce health hazards (McCutcheon and Schnoor, 2003). A major challenge is that
most tailing deposits cannot serve as a substrate for most plant
species because of metal toxicity, low pH, lack of essential minerals,
lack of clay and organic matter to retain water, lack of soil structure,
or lack of a seed source from nearby native plants or some combination of these factors (Mendez and Maier, 2008). Tailings can
remain devoid of plants for many decades or, in some cases, have
only slight plant cover (Gonzalez-Chavez et al., 2009). One approach
to modify the inhospitable substrate into a soil-like status can be
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accomplished by adding large quantities of compost, biosolids, and
water (Ye et al., 2001; Chiu et al., 2006). Though theoretically feasible, amendments become costly if applied to extensive areas with
tailings. Frequently, these sites are remote and water and irrigation
facilities are absent, especially at long-abandoned mine sites.
A more practical alternative is to use pioneering plants, mainly
leguminous shrubs and trees that fix nitrogen. This initial stage
improves soil characteristics by adding organic content and may
reduce soil toxicity. Later, more sensitive plants may grow; this creates a more stable and diversified ecosystem. One suitable pioneer
legume is the genus Sesbania, which tolerates detrimental effects
of heavy metals and supplies much-needed nutrients to the soil
as it decomposes, especially nitrogen (Chan et al., 2003). Inoculation with PGPB and also AM fungi has been proposed to support
cultivation of plants on tailings using smaller amounts of compost
(Mendez and Maier, 2008; Petrisor et al., 2004; Solís-Domínguez
et al., 2011; Zhuang et al., 2007) (Fig. 6).
There are two approaches in the search for PGPB compatible
with mine tailings. One is to isolate native PGPB from tailings or
from plants already growing on tailings, culture the bacteria, and
use them as inoculants. This is a labor intensive procedure that
has been successfully tested (Grandlic et al., 2008, 2009). This
approach assumes that the PGPB isolated from tailings provide
better support for plant survival and growth through several mechanisms, including increasing availability of nutrients, increasing
resistance to metal toxicity, or decreasing bioavailability of toxic
metals in the rhizosphere (Burd et al., 1998, 2000; Pishchik et al.,
2002; Glick, 2003; Belimov et al., 2004; Reed and Glick, 2005; Reed
et al., 2005; Vivas et al., 2006; Wu et al., 2006a; Li et al., 2007;
Rajkumar and Freitas, 2008a,b). Another approach is to test wellstudied PGPB used in other applications, mainly agriculture, that
could help plants grow on mine tailings. For example, strains of
Bacillus enhance growth of agricultural crops, wild plants, trees,
microalgae, and test plants by means of different mechanisms of
plant growth (Bashan et al., 2000b; Enebak et al., 1998; Hernandez
et al., 2009; Kloepper et al., 2004a,b; Ryu et al., 2005; Vessey,
2003). Also, strains of Bacillus are found in mine tailings (Natarajan,
1998; Vijayalakshmi and Raichur, 2003; Wu et al., 2006b; Tsuruta,
2007). The genus Azospirillum is commonly used for its growthpromoting activities and is probably the best studied of the PGPB,
except for rhizobia (Bashan and de-Bashan, 2010). Root stimulation
and reduced salt stress are common concerns in phytostabilization of mine tailings in semi-arid and arid ecosystems. Stimulation
of adventitious root growth by A. brasilense enhances nutrient
uptake, but also alleviates salt stress in agricultural plants (Bashan
et al., 1999; Bacilio et al., 2004; Creus et al., 1997; Dimkpa et al.,
2009). This stimulatory action provides increased surface area for
absorbing nutrients in the highly impacted, low-nutrient deposits
of tailings. Increased root mass also adds physical stability against
wind and water erosion. In addition to promoting plant growth
and enhancing resistance to abiotic stress, properties of Azospirillum that are relevant for their use include the ability to grow under
extremes of pH (de-Bashan et al., 2005) and heavy metals (Belimov
and Dietz, 2000).
Another alternative available with legumes is inoculation with
rhizobia. Several species of Sesbania were tested for remediation
of tailings containing lead, zinc, and copper. Inoculated plants
generally produced a higher biomass than plants that were not
inoculation (Chan et al., 2003). Yellow lupine L. luteus was tested
for in situ reclamation of multi-metal-contaminated soil after a
mine water spill. It accumulated heavy metals mainly in roots;
however, lead, copper, and cadmium were not well translocated
to shoots. This demonstrated that yellow lupine is potentially useful in metal phytostabilization. Inoculating lupines with a mix of
metal-tolerant PGPB (Bradyrhizobium spp., Pseudomonas spp., and
Ochrobactrum cytisi) produced further increase in biomass. At the
same time, metal accumulation in shoots and roots declined, which
may have resulted from a protective effect that the PGPB exerted
on the plant rhizosphere (Dary et al., 2010).
While there is a similarity in the concepts of phytostabilization and revegetation of severely eroded areas mentioned earlier,
the main differences involve presence of toxic metals and sometimes extreme pH of the tailings. Several studies demonstrated
the benefits of using native PGPB to enhance plant growth in phytostabilization projects (Grandlic et al., 2008, 2009; Petrisor et al.,
2004; Vivas et al., 2006). These PGPB can significantly increase root
and shoot length, biomass, and improve overall plant nutrition,
despite the presence of heavy metals. These fairly new studies propose mechanisms used by bacteria to improve vegetative growth,
similar to mechanisms of PGPB employed in agriculture: (1) PGPB
with ACC-deaminase activity stimulate root elongation and general plant growth in soils containing cadmium, but do not enhance
extraction of metals (Belimov et al., 2001); (2) PGPB alleviate heavy
metal toxicity by reducing sensitivity of plants to higher concentrations of cadmium, copper, zinc, and nickel, yet reduce absorption
of the metals by the plants (Belimov et al., 2004; Burd et al., 1998;
Reed et al., 2005; Vivas et al., 2006).
Several studies demonstrate the feasibility of using PGPB for
phytostabilization; some were done in combination with compost.
Some studies show that the optimal quantity of compost for establishment of several species of grasses and plants on tailings can be
reduced by using PGPB that were isolated from tailings or areas near
tailings (Grandlic et al., 2008; Petrisor et al., 2004; Zhuang et al.,
2007). In one study, chemical fertilizer was replaced with repeated
inoculations of Azotobacter chroococcum and Bacillus megaterium;
this significantly enhanced the growth of native species and microbial activity in two acidic tailing sites (Petrisor et al., 2004). Several
studies used desert shrubs and trees to demonstrate the capacity of native and commercial PGPB to improve growth of several
types of mine tailings. PGPB isolated from these tailing survived
strong acidic pH and high concentrations of heavy metals (Mendez
et al., 2008). When moderate amounts of compost was added with
the PGPB (Iverson and Maier, 2009), biomass of desert quailbush
Atriplex lentiformis and buffelgrass Buchloe dactyloides significantly
increased. Inoculating seeds of these plants with PGPB prior to
planting resulted in significantly increased dry biomass of native
desert plants raised on sulfurous tailings, some even without compost. The effect was partly dependent on the amount of the compost
amendment (Grandlic et al., 2008, 2009; Iverson and Maier, 2009).
Similarly, when quailbush was inoculated with B. pumilus isolated from roots of cacti growing on soilless rocks in an extreme
environment, the bacterium significantly improved the growth of
quailbush with a smaller amount of compost (10% w/w compared
to 15% normally used on these tailings). This treatment markedly
affected the bacterial community of acidic tailings with high metal
content and with neutral tailings with low metal content (deBashan et al., 2010a). Using several strains of A. brasilense used
in agricultural applications caused similar effects on plant growth
and the microbial community in acidic, metalliferous tailings (deBashan et al., 2010a,b). Inoculation of buffelgrass with a mix of
two PGPB (Arthrobacter spp.) changed the structure of rhizosphere
bacterial communities; after 75 days, the inoculants had increased
growth of buffelgrass (Grandlic et al., 2009).
There are at least two plausible explanations for the effect
of inoculation on bacterial communities in tailings that are not
apparent when these bacteria are added to agricultural soils.
Tailings usually have extremely low content of heterotrophic
bacteria (<103 CFU g−1 ) (Mendez et al., 2007). With a relatively
small community of bacteria, inoculation of either B. pumilus
or A. brasilense (∼106 CFU g−1 ) creates a large shift in the composition of native bacteria. Additionally, PGPB have an indirect,
but long-term effect on the native bacterial population because
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183
Fig. 6. Proposed process of phytostabilization of mine tailings.
the inoculum influences root growth or plant exudates after the
initial population of PGPB declines (de-Bashan et al., 2010a,b).
Long-term influence (5 years) on growth of cacti and desert
leguminous trees in impoverished soils occurred, even when the
initial inoculum had disappeared years earlier (Bashan et al., 1999,
unpublished).
Because some leguminous trees tolerate soils that are contaminated with heavy metals (Senthilkumar et al., 2005), they are
candidates for revegetation of tailings. Inoculation with Bradyrhizobium sp. of the leguminous lebbeck tree Albizia lebbeck significantly
increased nodulation and various growth parameters grown on
gypsum and limestone tailings (Rao and Tak, 2001). Although inoculation sometimes enhances metal uptake in mesquite, this did not
happen when several AM fungi were inoculated onto mesquite
growing on acidic tailings containing high levels of heavy metals (Solís-Domínguez et al., 2011). Inoculation with a combination
of the PGPB A. brasilense, AM fungi, and compost significantly
enhances growth of seedlings of the mesquite amargo on phosphate mine tailings (Moreno et al., unpublished data) similar to the
effect of AM fungi on this tree.
The end goal of phytostabilization, as part of general phytoremediation, is not only to establish a vegetative cap to stabilize metal
contaminants in the root zone, but to modify the soil to enhance
plant succession, that is, support a diversity of native plants close
to the natural vegetation of local soils (Fig. 6). Ideally, a transition
would include a more diverse indigenous microbial community,
one capable of supporting plant growth and nutrition. Inoculation
with PGPB used in agriculture enhances plant growth when supplemented with compost and enhances continued ecological impacts
on the rhizosphere microbial community. These are distinctly different from the microbial community residing on uninoculated
plants. Also, plants that have not been inoculated have less robust
aerial and root growth.
A potential negative impact of using PGPB for phytostabilizing mine tailings occurs when toxic metals accumulate in aerial
parts of plants, rendering the plants toxic to grazing animals. For
example, roots of Indian mustard B. juncea inoculated with PGPB
had minor absorption of copper, lead, and zinc, but significantly
increased cadmium uptake in shoots (Wu et al., 2006a,b). Copper,
cadmium, manganese, and zinc increased in shoots of native plants
after inoculations (Petrisor et al., 2004). In still another study, after
inoculation, concentrations of heavy metals accumulated in shoots
of canola, which can happen when canola is grown on soils with
low concentrations of metal. The low concentration was tolerated
by canola (Reed and Glick, 2005).
In summary, for hostile environments, such as toxic mine tailings, an inoculant PGPB can not only persist and stimulate plant
growth but directly or indirectly influence development of rhizobacterial communities. A PGPB can be native to the tailings
or a common agricultural inoculant. PGPB can be a component
of phytostabilization programs, together with compost and suitable plants that do not accumulate metals in their shoots to
avoid poisoning grazing animals. This is the most viable option
for achieving coverage of toxic tailings. Part of this process
is to understand the relationship between PGPB and organic
amendments or find plant–PGPB combinations that do not need
compost.
2.9. PGPB to improve tertiary wastewater treatment
Tertiary treatment of wastewater is done mainly by chemical
precipitation and, to a lesser extent, by biological means, such
as bacteria and microalgae, but only to a limited extent because
most wastewater only receives secondary treatment (digestion of
organic matter) before it is discharged or sold for irrigation (deBashan and Bashan, 2010). Developing efficient and cost-effective
treatments have major significance because common contaminants are increasing. Most wastewater treatment facilities will
face these issues in the near future. Numerous species of microalgae are commonly used to remove nitrogen and phosphorus
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in municipal wastewater (Tam and Wong, 2000; Valderrama
et al., 2002). PGPB are not employed in current technologies.
The idea of using PGPB for wastewater treatment came from
testing if microalgae respond to PGPB application in ways similar to higher plants. Because microalgae are basically single-cell
plants, as plants they may respond to PGPB application as do
other plants. The concept of an association of the microalgae
Chlorella vulgaris and the PGPB A. brasilense in alginate beads was
developed as part of the phycoremediation approach (de-Bashan
and Bashan, 2008; Gonzalez and Bashan, 2000). This combination is highly efficient at removing nitrogen and phosphorus
from several types of wastewater to prevent algal blooms, leaves
water cleaner, and provides a waste of usable biomass (de-Bashan
et al., 2002a, 2004). When immobilization of two microorganisms is undertaken (also known as co-immobilization), microalgae
remove contaminants and the PGPB enhances its capacity to
absorb these nutrients by changing metabolism and proliferation.
The effectiveness of using the microalgae C. vulgaris and Chlorella
sorokiniana immobilized with the well-known PGPB A. brasilense
in alginate beads under diverse environmental conditions was
intensively studied in recent years (de-Bashan et al., 2002a,b,
2004, 2005, 2008a, 2011; Gonzalez and Bashan, 2000; Lebsky
et al., 2001) (Fig. 7). A few other studies have also demonstrated
that other bacterial genera, including Rhodanobacter, Pseudomonas,
Bacillus, and Paenibacillus may advance wastewater treatment
(Hernandez et al., 2009; Mouget et al., 1995; Ogbonna et al.,
2000).
Early studies showed that A. brasilense increased growth rate,
population size, and lifespan of C. vulgaris by delaying senescence
(Gonzalez and Bashan, 2000; Lebsky et al., 2001). By immobilizing this microalga with A. brasilense, almost all ammonium and
about one-third of the phosphorus was removed after 2 days
of incubation in a batch culture, compared to using only C. vulgaris cells (de-Bashan et al., 2002a). A. brasilense has beneficial
effects on parameters of microalgae growth, including increased
production of pigments, lipid content, changing pattern of appearance of fatty acids in the cells, increased number of microalgal
cells, and sometimes, larger cell size (de-Bashan et al., 2002b).
A. brasilense Cd consistently enhanced growth of C. sorokiniana
or C. vulgaris, removed ammonium from wastewater, and slightly
reduced nitrates (de-Bashan et al., 2004).
To improve synergism between the two microorganisms, several environmental factors that affect growth of microalgae and
its cellular mechanisms were explored. These factors included pH,
concentration of carbon and nitrogen (affects nitrogen uptake in C.
vulgaris cells), light intensity at high temperature, and presence of
tryptophan. Hormonal effects were also evaluated (de-Bashan and
Bashan, 2008; de-Bashan et al., 2005, 2008a,c). A. brasilense alleviated tryptophan toxicity and extreme pH (de-Bashan and Bashan,
2008; de-Bashan et al., 2005). These physiological studies indicate
that a plausible mechanisms controlling growth is production of
the phytohormone IAA and enhancement of two key enzymes (glutamate dehydrogenase and glutamine synthetase) that affect the
nitrogen cycle and are also responsible for absorbing ammonium
into the microalga (de-Bashan et al., 2008b).
Starvation periods have a profound effect on assimilating
phosphorus from wastewater by C. vulgaris and C. sorokiniana
immobilized with A. brasilense. Joint immobilization enhanced
growth and absorption of phosphorus in both microalgae in synthetic wastewater and in C. sorokiniana in municipal wastewater
following a 3-day starvation in a saline solution. Phosphorus
removal did not increase after a starvation period and return of
the microalgae to the same wastewater; however, if cells were
returned to a new batch of wastewater, removal of phosphorus
continued. The most effective removal of phosphorus (72%), about
Fig. 7. PGPB (Azospirillum spp.) that are jointly immobilized with microalgae (Chlorella spp.) in alginate beads to improve tertiary wastewater treatment under authotrophic
and heterotrophic conditions. Abbreviations: Azo = Azospirillum spp., Ch = Chlorella spp., GS = glutamine synthetase, GDH = glutamate dehydrogenase, IAA = indole-3-acetic
acid, P-ase = phosphatase.
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double the rate of common removal of phosphorus by suspended
bacteria or microalgae, was achieved when starved, immobilized
cells of microalgae and bacteria were used for treatment for one
cycle and replaced with a fresh, starved culture (Hernandez et al.,
2006). When cultivation was changed to heterotrophic conditions
(without light and with an additional simple carbon source, glucose or acetate), the system of microalgae and bacteria performed
better (Perez-Garcia et al., 2010). To improve fertility of desert
soils containing very little organic matter; the dried debris of alginate beads containing C. sorokiniana and A. brasilense from tertiary
treatment of wastewater was used as an amendment. Three applications of debris increased organic matter, organic carbon, and
microbial carbon in the soil. Sorghum grew better in the amended
soil than sorghum grown in unfertile, untreated soil (Trejo et al., in
press).
The advantage of this technology is that immobilized cells of
microalgae with bacteria are always more effective at removing nitrogen and phosphorus than microalgae without bacteria.
As the two microorganisms are immobilized in alginate beads
that are easily and rapidly removed from wastewater by sedimentation, this technology could be a cost-effective alternative
to chemical precipitation used to treat wastewater. It solves two
problems in standard microalgal technology, increasing the population of microalgae to a level sufficient to clean the wastewater,
and using the waste biomass when the cleaning process is completed.
185
species tested so far are not effective on enhancing plant growth
without adding organic matter.
(6) Immobilization of PGPB with microalgae in small polymer
spheres (beads) was effective in bioremediation of wastewater.
There is still no example of scaling up of this procedure.
A few general future prospects can be suggested for PGPB technology. PGPB can be developed for sustainable production of biofuel
crops in conjunction with phytoremediation of contaminated soils
and water or to improve revegetation and sustainable feedstock
production on marginal lands. With growing knowledge of complete genome sequences for many PGPB, exploitation of these data
should center on functional analysis of bioremediation questions
involving the genes, proteins, and metabolites in the PGPB. In summary, PGPB has major potential in bioremediation problems, but
the past efforts are only the beginning.
Acknowledgments
This review was mainly supported by the Consejo Nacional de
Ciencia y Tecnología (CONACYT grant 130656) and the Secretaria de
Medio Ambiente y Recursos Naturales, (SEMARNAT grant 23510).
Time for writing was provided by The Bashan Foundation, USA. Ira
Fogel of CIBNOR provided editorial services.
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