Bioresource Technology 98 (2007) 35353546

Agro-industrial waste materials and wastewater sludge for rhizobial

inoculant production: A review
F. Ben Rebah a, D. Prvost b,
, A. Yezza c, R.D. Tyagi c

a
INSTM-Sfax B.P. 1035, Sfax 3018, Tunisia
b
Agriculture et Agroalimentaire Canada, Sainte-Foy, QC, Canada G1V 2J3
c
INRS-Eau, Terre et Environnement (INRS-ETE), 490 rue de la Couronne, Qubec, QC, Canada G1K 9A9

Received 4 June 2005; received in revised form 12 November 2006; accepted 12 November 2006
Available online 2 March 2007

Abstract

Inoculating legumes with commercial rhizobial inoculants is a common agriculture practice. Generally, inoculants are sold in liquid or
in solid forms (mixed with carrier). The production of inoculants involves a step in which a high number of cells are produced, followed
by the product formulation. This process is largely governed by the cost related to the medium used for rhizobial growth and by the avail-
ability of a carrier source (peat) for production of solid inoculant. Some industrial and agricultural by-products (e.g. cheese whey, malt
sprouts) contain growth factors such as nitrogen and carbon, which can support growth of rhizobia. Other agro-industrial wastes (e.g.
plant compost, Wltermud, Xy-ash) can be used as a carrier for rhizobial inoculant. More recently, wastewater sludge, a worldwide recycla-
ble waste, has shown good potential for inoculant production as a growth medium and as a carrier (dehydrated sludge). Sludge usually
contains nutrient elements at concentrations suYcient to sustain rhizobial growth and heavy metals are usually below the recommended
level. In some cases, growth conditions can be optimized by a sludge pre-treatment or by the addition of nutrients. Inoculants produced in
wastewater sludge are eYcient for nodulation and nitrogen Wxation with legumes as compared to standard inoculants. This new approach
described in this review oVers a safe environmental alternative for both waste treatment/disposal and inoculant production.
2007 Elsevier Ltd. All rights reserved.

Keywords: Rhizobium; Inoculant; Raw material; Waste; Sludge

1. Introduction commercial formulations containing rhizobia that can be

The rhizobium-legume symbiosis plays an important
role in agriculture, because it oVers the ability to convert
atmospheric molecular nitrogen into forms useable by the
plant, a process called biological nitrogen Wxation. Most of
the research to optimize symbiotic nitrogen Wxation and to
increase the use of legumes in crops systems has been in
part stimulated by increasing fertilizer prices and by envi-
ronmental concerns. One beneWt of increased knowledge of
symbiotic nitrogen Wxation is the inoculation of legumes by
selected rhizobia, a practice that allows producers to intro-
duce rhizobia into the soilplant ecosystem. Inoculants are
*
Corresponding author. Tel.: +1 418 657 7980; fax: +1 418 648 2402.
E-mail address: prevostd@agr.gc.ca (D. Prvost).
applied to the seed or the soil during planting (reviewed by
Brockwell and Bottomley, 1995). It was estimated that
approximately 2000 tons of inoculants are produced
worldwide annually, a quantity suYcient to inoculate 20
million ha of legumes. The largest single producers are in
the USA with an annual production of 1000 tons (Single-
ton et al., 1997). Inoculants are usually commercialized as
solid inoculants, in powder from peat or in granular form,
or as liquid inoculants, in broth formulations (Stephens
and Rask, 2000). Alternative formulations such as oil
based products or concentrates, lyophilized cultures or rhi-
zobial cells entrapped in beads have been investigated. The
method of inoculation depends on the inoculant type: pow-
dered and liquid inoculants are generally applied to the
seeds, while granular inoculants are applied to the soil, in
the furrow.

0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.11.066
3536 F. Ben Rebah et al. / Bioresource Technology 98 (2007) 35353546
Solid inoculants are prepared by adding fermenter-
grown broth, containing a large population of rhizobium,
to a powdered carrier followed by a period of maturation
to ensure multiplication or adaptation of rhizobial cells
(Roughley and Pulsford, 1982). Thus, the production of a
large quantity (greater than 109 cells per mL) of rhizobia in
liquid medium constitutes the Wrst step in the production
of powdered legume inoculants. The economies of this pro-
cess are governed by the cost and availability of a suitable
carbon source (Bissonnette et al., 1986). The standard
medium, including mannitol as carbon source and yeast
extract as a source of nitrogen, growth factors and mineral
salts, has been used for laboratory scale production, but its
industrial use is limited due to high cost. Among carriers
that can sustain high levels of rhizobia, peat is the most
widely used (Burton, 1967; Peterson and Loynachan,
1981), but is not universally available (Tilak and Subba
Rao, 1978; Corby, 1976). Alternatively, diVerent materials
such as industry by-products, organic wastes, mineral soils,
plant by-products, coal, perlite, and agro-industrial wastes
have been tested as culture media for the growth of rhizo-
bia (Brockwell and Bottomley, 1995; Stephens and Rask,
2000). Most of the materials are local resources easily
available in a speciWc region. In this review we will present
the potential of diVerent products and wastes tested as
growth substrates and carriers for rhizobia. Emphasis will
be given to wastewater sludges which have been extensively
characterized and have proven to be very good material for
inoculants production.
2. Need for inoculation
Biological nitrogen Wxation by rhizobia represents
approximately 7080% of the total nitrogen accumulated
by legume plants. Estimates of nitrogen Wxation in legume
crops are 105 and 200 kg N ha1 yr1 for clover, 169 kg
N ha1 yr1 for lupine, 208 kg N ha1 yr1 for alfalfa and
234 kg N ha1 yr1 for soybean (Brockwell and Bottomley,
1995). This process has the advantage of reducing the
dependency of agriculture on nitrogen fertilizers, and it is
economical, using energy from photosynthesis. Fixed nitro-
gen is beneWcial to soil fertility and reduces the environ-
mental pollution associated with the use of nitrogen
fertilizers.
Several soil and environmental stresses aVect the perfor-
mance of the legume-rhizobium symbiosis and may limit
nodulation and nitrogen Wxation. Rhizobial populations,
the host plant species, agronomic practices and climatic
conditions are among the important factors. When a new
crop is introduced, rhizobia may be absent in soils or indig-
enous populations may be low due to unfavorable soil con-
ditions, especially low pH (Catroux et al., 2001). Since
rhizobia should be in suYcient numbers to ensure optimal
nodulation and eYcient nitrogen Wxation, inoculation of
legumes with high populations of speciWc and eYcient rhi-
zobial strains has proven to be a valuable agronomic strat-
egy to improve crop productivity.
3. Quality of commercial legume inoculants
Commercial inoculants have been used for more than a
century, and they have contributed to increased crop pro-
ductivity. A good inoculant must be prepared with a strain
of rhizobium selected for high N Wxation eYciency and
competitive ability for nodulation. The strain must survive
in the inoculant formulation, maintain its properties during
storage, and be tolerant to stress factors such as acidity,
desiccation, high temperature and chemical pesticides. The
most important factor for inoculant quality is a high num-
ber of live rhizobia (greater than 2 109 rhizobia per g),
and no or minimal contamination by microorganisms not
detrimental to rhizobia or pathogen to plants and humans
(Lupwayi et al., 2000).
Many countries, including Australia, the Netherlands,
Rwanda, and Thailand have regulated minimum standards
varying from 5 107 to 1 109 rhizobium cells/g of inoculant
product (Lupwayi et al., 2000). In Canada, the standards
stipulate a minimum number of rhizobia delivered per seed:
103 rhizobia per small seed (e.g. clover and alfalfa), 104 per
medium seed (e.g. mungbean, pigeonpea, sainfoin) and 105
per large seed (e.g. soybean, pea and bean). Thus, the number
of rhizobia required in an inoculant product depends on the
host legume species (seed size) and on the rate of application
of the product suggested by the manufacturer. For example,
for inoculant products sold in 1997 in Canada, the minimal
number of rhizobia required to meet the standards varied
from 2 106 to 1.3 109 rhizobium cells per g or mL product
(CFIA, 1997). France determined a minimum standard of
106 rhizobial cells per seed for soybean (Catroux, 1991)
because only a small portion of the applied inoculum is asso-
ciated with the seed after planting. In the United States and
the United Kingdom product quality and rates of applica-
tion are left to the discretion of the manufacturer (Smith,
1992). Shelf-life is also an important inoculant characteristic
for farmers and manufacturers. Inoculant manufacturers
prefer products with a shelf-life of 11.5 years under ware-
house conditions in order to be able to sell them over two
cropping seasons (Catroux et al., 2001). Many inoculants
produced with local alternative carriers can maintain accept-
able numbers of rhizobia for 312 months (see Section 5,
production of legume inoculants), which is suYcient if they
are used shortly after their production.
4. Nutrient requirements of rhizobia
Based on growth rate, nutritional requirement and
enzymes, rhizobia can be traditionally divided in two
groups: the fast-growing rhizobia, placed in the genus Rhi-
zobium and the slow-growing rhizobia represented by the
genus Bradyrhizobium. More recently, phenotypic and geno-
typic characterization of rhizobia isolated from an increas-
ing number of plant species has led to the reclassiWcation
and new designation of some species and to the description
of additional genera and species (Zakhia and De Lajudie,
2001; Sahgal and Johri, 2003). In this review, rhizobial spe-
 F. Ben Rebah et al. / Bioresource Technology 98 (2007) 35353546
cies commonly used in legume inoculants will be designated
according to the current taxonomy, although they were des-
ignated diVerently in many studies on inoculant production,
approximately 20 years ago. The fast-growing species are:
Sinorhizobium meliloti (formerly Rhizobium meliloti) for
alfalfa; Rhizobium leguminosarum bv. viciae (R. leguminosa-
rum) for peas, lens and vetches; bv. phaseoli (R. phaseoli) for
beans; bv. trifolii (R. trifolii) for clover; Rhizobium tropici
for beans and R. sp. (Cowpea) for cowpea. There is one spe-
cies of intermediate growth rate: Mesorhizobium loti (R. loti)
for birdsfoot trefoil. The slow-growing species are Brady-
rhizobium japonicum (R. japonicum) and B. elkanii for soy-
bean and B. sp. (Lupinus) for lupine.
Rhizobial species diVer in their physiological and bio-
chemical characteristics which inXuence their growth
requirements. This knowledge is useful in selecting organic
wastes and optimizing their properties (e.g. by adding nutri-
ents) for inoculant production of a given rhizobial species.
The fast-growing rhizobia (e.g. Rhizobium, Sinorhizobium)
are acid producers; they have a mean generation time of 2
4 h in standard yeast mannitol broth (YMB) and form colo-
nies of 24 mm diameter, after 35 days of incubation on
yeast mannitol agar (YMA) (Jordan, 1984). Slow-growing
rhizobia (Bradyrhizobium) are alkali-producers; they
exhibit a doubling time of 68 h and form colonies not
exceeding 1 mm in diameter after 57 days of incubation on
YM media (Jordan, 1984).
Fast- and slow-growing rhizobia also diVer in their
capacity to utilize carbohydrates. Fast-growing bacteria
can grow on a large variety of carbon substrates, such as
hexoses, pentoses, disaccharides, trisaccharides and organic
acids (Allen and Allen, 1950). Slow-growing rhizobia have
a more restricted range of carbon sources, they grow in the
presence of pentoses, and they can also utilize many aro-
matic substrates (Parke and Ornston, 1984).
All rhizobial species require nitrogen sources, vitamins
and trace minerals for growth. Some fast-growing rhizobia
can utilize nitrate, ammonia and amino acids as their sole
nitrogen source (Quispel, 1974). Optimal growth can be
achieved by adding low weight amino acids, but the balance
of amino acids is important (Burton, 1979). However, rhi-
zobia grown on media enriched with glycine, alanine and
certain D-forms of amino acids loose the capacity of nitro-
gen Wxation (Burton, 1979). Vitamin requirements vary
among rhizobial species; R. leguminosarum (bv. trifolii and
bv. phaseoli) needs biotin, thiamine or calcium pantothe-
nate or a combination of these three vitamins; S. meliloti,
B. japonicum, and other species need only biotin (Graham,
1963). Generally, vitamin included in yeast extract satisWes
needs of rhizobia.
Rhizobia also require mineral salts for growth. Yeast
extract contains signiWcant concentrations of salts (K, Ca,
Mg, and others) (Burton, 1979). Calcium deWciency aVects
rhizobial growth, and low concentrations (0.1 ppm) produce
abnormal cells (Vincent, 1962). InsuYcient quantities of Mg
cause an abnormal cell multiplication (Norris, 1959). Certain
trace minerals such as cobalt, zinc, manganese and iron are
3537
required at low concentrations for rhizobial growth (Lowe
and Evans, 1962; Wilson and Reisenauer, 1970a,b).
Yeast extract can be used as a sole carbon and nitrogen
source by rhizobia (Meade et al., 1985) since it contains,
amino acids, inorganic nitrogen and growth factors (Fe, Ca,
Mg, St, K, Ba, Mn, Cu, Pb, Al and Va) at a concentration
for rhizobial growth (Burton, 1979). The addition of yeast
extract to growth media improves the eYciency of rhizobial
growth (Meade et al., 1985; Bissonnette and Lalande, 1988;
Ben Rebah et al., 2002d), but high concentrations are not
economical for large industrial production. Concentrations
higher than 0.35% are not beneWcial because they produce
distorsed cells and decrease the viability of some rhizobial
strains (Skinner et al., 1977).
5. Production of legume inoculants
5.1. Quantity production of rhizobia in liquid cultures
5.1.1. Standard medium
The standard medium YMB used initially for laboratory
scale production of legume inoculant contains the follow-
ing constituents (in grams per liter): K2HPO4, 0.5;
MgSO4 7H2O, 0.2; NaCl, 0.1; yeast extract, 1; and manni-
tol, 10 (Vincent, 1970). It has been modiWed by introduction
of other carbon sources: sucrose is the most commonly
used carbon source, and mannitol and glycerol are
sometimes used for slow-growing rhizobia (Burton, 1979).
Glycerol has the most beneWcial eVect on growth of slow-
growing rhizobia (Arias and Martinez-Drets, 1976).
5.1.2. Agricultural and industrial by-products
Numerous investigators have looked for ways of pro-
ducing rhizobia using low cost media. A variety of agricul-
tural and industrial by-products contain growth factors,
carbon and/or nitrogen and oVer good potential as culture
media, as shown by the high quantity of rhizobial cells pro-
duced (Table 1).
Cheese whey: The by-product of the cheese industry was
used for culture of the fast-growing rhizobium, S. meliloti
(Bissonnette et al., 1986). Whey contains the majority of ele-
ments included in the standard media at high enough con-
centrations to allow rhizobial growth (Vincent, 1974).
According to Cerbulis et al. (1982), lactose in whey repre-
sents approximately 7075% of the total solids while nitro-
gen represents 1.822.4%. The lactose uptake system of S.
meliloti is inducible, and its catabolism involves cleavage to
monosaccharides by -galactosidase (Glenn and Dilworth,
1981; Bissonnette et al., 1986). To avoid any latency on whey,
rhizobium inoculum was produced under inducing condi-
tions. Hence, whey allowed rapid growth of S. meliloti and
gave a cell population similar to that obtained in YMB. In
whey media, stationary growth was reached after 48 h, and
the maximum cell number of 6.3 109 cfu/mL was obtained
within 72 h. Whey supplemented with yeast extract (1 g/L)
and phosphate (0.5 g/L) increased the cell number to
1 1010 cfu/mL after 48 h (Bissonnette et al., 1986). The same
3538 F. Ben Rebah et al. / Bioresource Technology 98 (2007) 35353546
Table 1
Production of biomass from diVerent species of rhizobia grown in agricultural and industrial by-products
Wastes Rhizobial strains
Whey S. meliloti
Malt sprouts S. meliloti
S. meliloti
R. leguminosarum bv. phaseoli
R. leguminosarum bv. viciae
B. japonicum
Grade yeast extract R. leguminosarum bv. viciae
Pea husk, molasses R. leguminosarum bv. trifolii
and water hyacinth R. leguminosarum bv. trifolii
B. japonicum
B. japonicum
B. japonicum and R. sp.
a
Growth time in parentheses.
authors demonstrated that whey protects rhizobial cells from
freezing and thawing damage, since S. meliloti grown in
whey survived freezing at 18 C better than cells grown on
mannitol or sucrose (Bissonnette and Lalande, 1988). More-
over, rhizobia produced in whey and stored in peat survived
better at various temperatures of storage as shown by 100%
survival after 23 weeks at 4 C. Therefore, the by-product
of the dairy industry oVers an interesting potential as growth
media and as protectant for industrial inoculants.
Malt sprouts: The by-product of the malt industry (malt
sprouts) was also used as a culture medium for rhizobia
(Boiardi and Ertola, 1985). Malt sprouts extract was pre-
pared by heating a mixture of malt sprouts and water at
100 C for 30 min. After centrifugation, a liquid was
obtained that contained 3.75% w/v of solids, 0.18% w/v of
nitrogen and 0.6% w/v of total reducing sugars. Many rhizo-
bial species (R. leguminosarum bv. phaseoli and bv. viciae, S.
meliloti, B. japonicum) were tested in media containing
diVerent concentrations of malt sprouts extract. The malt
sprouts extract at concentrations varying from 0 to 80% v/v
of the growth medium were used to replace yeast extract in
the medium containing: glycerol, yeast extract, peptone,
K2HPO4, KH2PO4, MgSO4 7H2O, FeCl3 6H2O, NaCl,
MnSO4 and biotin. In batch culture, all strains grew well in
the malt sprouts based medium and reached a concentration
higher than 5 109 cfu/mL. DiVerences between slow and
fast-growers were observed: cell yields of the fast-growing
rhizobia increased with increasing concentration of the malt
sprouts extract, while a concentration of 40% v/v inhibited
the growth of the slow-growing rhizobia (B. japonicum).
Industrial-grade yeast extract: With the aim to produce
cell concentrates of R. leguminosarum bv. viciae, Meade
et al. (1985) compared standard YMB with a medium using
industrial-grade yeast extract as the carbon and nitrogen
source under industrial-scale conditions. The growth yield
in yeast extract (5 g/L) medium was 2.4 109 and
8.1 109 cfu/mL for medium containing 0.6 and 0.9 g/L of
phosphate buVer respectively, and these values were similar
to that of 3 109 cfu/mL obtained in standard YMB
Type of culture vessel Max cell count (cells/mL) References
5-L fermentor (48 h)a 4.7 109 Bissonnette et al. (1986)
1-L Xask >6 10 9
Boiardi and Ertola (1985)
5-L fermentor >5 109
1-L Xask >3 109
1-L Xask >5 109
1-L Xask >5 109
200-L fermentor (40 h) 2.4 109 to 8.1 109 Meade et al. (1985)
25-L fermentor (72 h) 2.80 1010 Gulati (1979)
135-L fermentor (72 h) 1.75 1010
25-L fermentor (96 h) 2.20 1010
135-L fermentor (84 h) 1.75 1010
(144 h) 9.86 109 Jain et al. (2000)
medium. Yeast extract was advantageous for cell recovery
with a continuous centrifugation system since the recovery
was 63% and 70% in yeast extract medium in comparison
to only 0.6% in standard YMB medium. The better recov-
ery in yeast extract medium was attributed to the absence
of polysaccharides which, in the YMB medium, trap cell
within the viscous supernatant.
Pea husk, molasses and water hyacinth: Agriculture
wastes were used to formulate a non-synthetic medium
(Gulati, 1979) that contained pea husk, molasses, water
hyacinth, mineral salts (NaCl, CaCO3, K2HPO4, MgSO4)
and yeast extract. A mixture of pea husk and water hya-
cinth (sacchariWed with mycelium of Trichoderma viride)
was boiled for 4 h with 5% HCl and used to partially
replace the yeast extract normally present in standard
YMB. Many strains of R. leguminosarum bv. trifolii and B.
japonicum tested in Xask and in fermentors for large-scale
production showed equal growth in standard and in formu-
lated non-synthetic media. The suitability of the formulated
medium can be attributed to the presence of required
amino acids such as tryptophan, glutamic acid and carbon
sources such as glucose, arabinose and cellobiose. The
large-scale production (25 and 135 L fermentors) was ade-
quate for commercial use.
Jaggery: Jaggery is a coarse brown Indian sugar made
from palm-sap that has been tested as sole source of carbon
for three strains of B. japonicum and three strains of Rhizo-
bium sp. (Jain et al., 2000). In general, the number of cells
obtained in jaggery was similar to that obtained in manni-
tol or glycerol media, but a few strains grew faster and gave
a higher number of cells in jaggery.
5.2. Alternative carriers for production of solid inoculants
Most inoculants on the market are prepared in powdered
organic carriers such as peat by adding large populations of
rhizobia produced in broth to the carrier. The level of rhizo-
bia should also meet the required standards of quality vary-
ing from 107 to 109 cells/g depending on the country
 F. Ben Rebah et al. / Bioresource Technology 98 (2007) 35353546
(Lupwayi et al., 2000). Therefore, a carrier should be high in
organic matter with optimum nitrogen content, a pH
around neutral and a high water holding capacity. It should
be non-toxic, permit growth after incubation, be available
all year-round and be inexpensive. Peat is the most widely
used carrier, and it can sustain high numbers of rhizobia
(greater than 108 cells/g) during storage at a temperature
range from 3 to 28 C (Burton, 1967; Van Schreven, 1970).
Granular preparations using peat prill or mineral granules
are also suitable for inoculation of diVerent farming systems
(Stephens and Rask, 2000). Since peat is not available in a
number of countries, particularly in regions without naturals
deposits of peat, alternative solid materials have been evalu-
ated as inoculants carriers (Brockwell and Bottomley, 1995).
They can be grouped into soil-based carriers, inert material,
and wastes from plant by-products and industry. The major-
ity of carriers are naturally abundant resources or available
waste materials. Viability of rhizobia is the most important
factor, and data for some carriers are listed in Table 2.
It is rather diYcult to compare results from diVerent
experiments using diVerent storage conditions. As seen in
Table 2, the storage temperature was either cold (4 C or
10 C) or ambient (20, 25 or 30 C), and the length of stor-
age varied from 24 days to 12 months. Despite of these
varying conditions, between 107 and 108 cells/g from diVer-
ent rhizobial species survived in most carrier materials,
which is acceptable to meet the standard required for qual-
ity inoculants. Among soil-based carriers, volcanic pumice
(Einarsson et al., 1993) oVered good potential for long term
storage (35 weeks) with populations higher than 108 cells/g,
while only 106 cells/g were observed in mineral soils after 15
weeks (Chao and Alexander, 1984). All plant materials sus-
tained populations varying from 107 to 108 cells/g except
cellulose powder which only sustained 106 cells/g after 24
days (Pugashetti et al., 1971). In general, inert material
showed a better potential than plant material. In polyvinyl-
pyrrolidone (PVP), populations were higher than 109 cells/g
(Denardin and Freire, 2000) and 108109 cells/g survived in
vermiculite (Graham-Weiss et al., 1987), perlite (Daza et al.,
2000) and bentonite (Choi et al., 1998). Rhizobia entrapped
in beads of alginate (Jung et al., 1982) seem to survive bet-
ter than those entrapped in polyacrylamide (Dommergues
et al., 1979). Recently, local agro-industrial wastes such as
oxalic acid production waste (Ajay et al., 1996) and Xy-ash
(Ghosh et al., 1996) have been successfully used as carriers
for the production of rhizobia. The alternative carriers have
the ability to sustain populations of rhizobia but in consis-
tent supply and quality, as well as cost limit their wide-
spread adoption (Stephens and Rask, 2000).
6. A new strategy: the use of municipal and industrial
wastewater sludge
6.1. Characteristics and recycling of wastewater sludge
Pollutants in industrial and municipal wastewaters can
be eliminated by physical, chemical and or biological treat-
3539
ment processes. These treatments generate sludge, which is
considered recyclable waste. Sludge is a complex suspen-
sion containing water (between 95% and 98%), organic and
inorganic matter and a large variety of microorganisms.
Sludge is classiWed into primary, secondary or digested
sludge depending on the nature of the treatment process,
the aZuent origin (municipal or industrial) and the rate of
pollutant elimination (Roques, 1979).
The province of Quebec (Canada) generated 218,000
tons of sludge (dry weight) in 2002 (Hbert, 2004). The
USA generated 6.9 million tons in 1998 and that is pro-
jected to increase to 8.2 million tons by 2010 (EPA, 1999).
Sludge disposal is an increasing environmental problem
and may be onerous; treatment and handling alone repre-
senting 50% of wastewater utility costs (Leblanc, 2005).
Land Wlling (EPA, 1993), ocean discharge (Gross, 1993),
conversion into useful materials such as oils (Campbell and
Martinoli, 1991) and building materials (Tay and Show,
1992) are some of the approaches used for sludge disposal
and recycling. Recently, emphasis has been placed on the
bioconversion of sludge into value-added products such as
biopesticides (Lachhab et al., 2001), enzymes (Tyagi et al.,
2002) and bioplastics (Ben Rebah et al., 2002a). The next
sections of this review will present evidences showing that
wastewater sludge also constitutes an eVective alternative
to produce rhizobium for legume inoculants.
6.2. Sludge as a growth media
6.2.1. EVects of sludge composition on rhizobial growth
Generally, the organic and mineral composition of
sludge varies depending on the origin and the treatment
process. However, sludges generated by municipal and
industrial wastewater treatment processes (Canada) used
by Ben Rebah et al. (2002b,d) contained suYcient carbon,
nitrogen, phosphorus and micronutrients to sustain bacte-
rial growth. Two major concerns associated with the use of
wastewater sludge as a sole raw material are the presence of
toxic heavy metals and pathogens. For pathogen removal,
the sludge is sterilized before rhizobial inoculation. As far
as heavy metals are concerned, many countries have regula-
tions for maximal metal concentration in sludge for agricul-
ture, forestry, and other uses. In the USA, sludge can be
classiWed as Class A, B or C according to the Environmen-
tal Protection Agency (EPA, 1993). Class A sludge can be
safely used in production of bacterial inoculant (Yezza
et al., 2005). In all types of sludges studied for rhizobial
inoculant production (Ben Rebah et al., 2002b,d), the con-
centrations of Cu, Zn, Cd, Ni and Pb were below the per-
mitted levels in the province of Quebec (Canada) and in the
European Economic Community. It is however worthy to
mention that heavy metals can inhibit the growth of rhizo-
bia, depending of their concentration (Wilson and Reis-
enauer, 1970a,b). Moreover, long exposure to metals
derived from past applications of sludges in soils may aVect
survival of eVective rhizobia, and consequently nitrogen
Wxation (McGrath et al., 1988; Giller et al., 1989). Zn and
3540 F. Ben Rebah et al. / Bioresource Technology 98 (2007) 35353546

Table 2
Alternative carriers used for solid-based-rhizobial inoculant
Carrier tested Rhizobial species Viability cells per g or mL or per seed References
Soil-based carriers
Mineral soils R. leguminosarum bv. phaseoli 106 at 4 C and 105 at 25 C, 105 d Chao and
Viability on coated seeds: mineral soil > peat Alexander (1984)
Coal (8 types) R. leguminosarum bv. phaseoli 107 to 1010 at 20 22 C, 4 wks Crawford and
Viability on coated seeds: coal 104 < peat 105 Berryhill (1983)
Volcanic pumice Bradyrhizobium sp. (Lupinus) 108109 at 22 C, 35 wks Einarsson et al. (1993)
(nutrient supplemented)
Inert material-based carriers
Vermiculite B. japonicum, S. meliloti and 108109 at ambient T, 4 wks (by direct Graham-Weiss et al. (1987)
(nutrient supplemented) R. leguminosarum bv. phaseoli fermentation of initial inoculum)
Bentonite + vermiculite S. meliloti 108 at 4 C, 12 mo Choi et al. (1998)
Polyacrylamide gel beads B. japonicum 7 107 at 4 C, 75 d (Peat: 1.5 109) Dommergues et al. (1979).
6.1 107 at 30 C, 75 d (Peat: 1 106)
(1) Alginate beads B. japonicum (1) 108 at 28 C, 90 d Jung et al. (1982)
(2) Xanthan and carob gum beads (2) 108 at 28 C, 300 d
Polyvinylpyrrolidone B. japonicum >109 at ambient T, 8 mo Denardin and
(PVP), xanthan, Jatai gum (carob) Freire (2000)
Perlite R. leguminosarum bv. phaseoli, Similar to peat at 4C, 6mo Daza et al. (2000)
R. tropici and B. japonicum Better than peat at 28C, 6mo
On coated seeds: similar to peat
with sucrose as adhesive, but not
with gum arabic
Agro-industrial wastes-based-carriers
Soybean meal and coir dust R. leguminosarum bv. trifolii Soil + soybean meal: 10 107; Iswaran (1972)
(added to soil) Soil + coir dust: 16 106; Soil: 7 107
Rice husk R.leguminosarum bv. trifolii and bv. 1.151.2 107 at 2831 C, 12 wks Khatri et al. (1973)
viciae, S. meliloti and B. japonicum
Cellulose powder B. japonicum 2 106 at 10 C and 1 106 at 30 C, 24 d Pugashetti et al. (1971)
Plant compost R. leguminosarum bv. trifolii and 4.57.2 109 at 26 C, 8 wks Iswaran et al. (1973)
B. japonicum
Sawdust (composted) (1) B. japonicum (1) 13.9 108 at 69 C, 9 mo Kostov and Lynch (1998)
(2) S. meliloti (2) 15 109 at 69 C, 9 mo
(3) M. loti (3) 7 109 at 69 C, 9 mo
Teak leaf meal, compost, charcoal, B. japonicum 1.78.5 109 at 30 C, 15 d (comparable Chakarapani and
Indian peat, lignite and mixtures to peat: 8.2 109) Tilak (1974)
(1) Sugarcane bagasse, sawdust Rhizobium sp. (Sesbania) (1) 105106 at 30 C, 90 d Muniruzzaman and
(2) Filter mud (2) 107 at 30 C, 90 d Khan (1992)
Sugarcane press mud B. japonicum 3 106 at 30C, 45 d Singh et al. (1992)
(alone or mixed)
Filter mud R. leguminosarum bv. trifolii and 108109 at ambiant T, 12 wks (air dried, Philpotts (1976)
R. sp. (Cowpea) unsterile and autoclaved)
Sugar mills B. japonicum 2.67.2 108 at ambient T, 3 mo Marafu et al. (1995)
7 9
Cotton stalks, groundnut shells, B. japonicum 1.2 10 2.2 10 at ambient T, 3 mo Potdukhe and
jowar stubbles (degraded) Somani (1997)
(1) Charcoal, vermiculite R. leguminosarum bv. phaseoli (1) similar to peat at 4 C and 25 C, 30 wks Sparrow and Ham (1983)
(2) Peanut hulls, corn cobs (2) not suitable
Fly-ash Rhizobium sp. (Leucaena, Albizia, 1.42.4 109 at ambient T, 3 mo Ghosh et al. (1996)
Acacia, Sesbenia, Dalbergia) 1.610.2 107 at ambient T, 12 mo
Oxalic acid industrial B. japonicum 109 at room T, 90 d Ajay et al. (1996)
waste (OIW)

Cd are the most toxic metals on survival of R. leguminosa- unlikely that rhizobia grown in sludges will be severely
rum bv. trifolii, but toxicity was observed only after eight aVected by heavy metals because sludge inhibitory factors
months at concentrations of 1.3 and 2.4 times the United are generally complexed by the organic matter. Moreover,
Kingdom limits (Chaudri et al., 1992, 1993). Therefore, it is the low inoculation rate of legume seeds (23 mL or 36 g
 F. Ben Rebah et al. / Bioresource Technology 98 (2007) 35353546 3541

of inoculant per kg of seeds) will introduce only very small
quantities of heavy metals in soils.
Ben Rebah et al. (2002b) demonstrated for the Wrst time
that sludge generated by industrial and municipal wastewa-
ter treatment processes sustains growth of various strains
of rhizobia (Table 3). Fast-growing rhizobia (Rhizobium,
Sinorhizobium) grew well in most sludge types, with popula-
tion higher than 1 109 cfu/mL, similar to that obtained
with standard YMB. In a few sludges (e.g. Valcartier pri-
mary sludge and the mixed sludge Quebec City), growth of
R. leguminosarum bv. viciae was inhibited and growth of S.
meliloti was reduced to 108 cfu/ml. Population levels of
slow-growing rhizobia (Bradyrhizobium) were often lower
(108109 cfu/ml) than those of fast-growing species and
lower than those obtained in standard YMB medium. Both
B. japonicum and B. elkanii could not grow in the Valcartier
primary sludge nor in the mixed sludge, indicating a lack of
available nutrient requirements or a toxicity eVect.
Therefore, rhizobial growth depends on the species and
on the sludge characteristics that aVect the growth time and
the cell yield. The Ca/Mg ratio seems to have more impact
on growth of rhizobia than the quantity of Ca and Mg sep-
arately (Vincent, 1962). For example, R. leguminosarum bv.
trifolii was not aVected by a Ca/Mg ratio that ranged
between 10 and 1/30, while S. meliloti grew better under
lower ratio conditions (Steinborn and Roughley, 1975). In
studies with wastewater sludges, the two most inhibitory
sludges (Table 3) had a low (2.13, Valcartier primary
sludge) and a high (6.7, Quebec city mixed sludge) Ca/Mg
ratio, suggesting that growth in sludge result from com-
plexes interactions.
6.2.2. EVects of sludge solids concentration on rhizobial
growth
As shown in Table 4, the concentration of suspended
solids in sludge aVected the growth of S. meliloti in primary
and secondary sludges from municipal wastewater treat-
ment plant (Quebec, Canada) (Ben Rebah et al., 2001,
2002b). Because rhizobia are aerobic bacteria, the high sol-
ids concentration (3.2%) found in original primary sludge
(Black Lake) could reduce oxygen diVusion and conse-
quently reduce cell growth to 1/10th of the growth in the
same sludge diluted to 1.3% solid concentration. Reducing
the solids concentrations by dilution of the original sludge

Table 3
Growth of diVerent rhizobial species in sludge tested for inoculant production (adapted from Ben Rebah et al. (2001, 2002a,b,c,d,e) and Dufresne (2004))
Sludge type Origin and process Strains tested Maximum cell count Generation
(cfu/mL) time (h)
Primary sludge Municipal wastewater plant, S. meliloti 1.12 109 10.22
Black Lake (Quebec) R. leg. bv. viciae 3.10 109 7.50
B. japonicum 0.67 109 8.82
B. elkanii 1.20 109 11.37
Municipal wastewater plant, S. meliloti 0.70 109 11
Valcartier (Quebec) R. leg. bv. viciae n.g. n.g.
B. japonicum n.g. n.g.
B. elkanii n.g. n.g.
Secondary sludge Municipal wastewater plant, S. meliloti 2.20 109 7.70
Black Lake (Quebec), sequential R. leg. bv. viciae 2.96 109 4.48
batch reactor B. japonicum 0.47 109 11.79
B. elkanii 0.50 109 10.58
Municipal wastewater plant, S. meliloti 2.45 109 6.52
Valcartier (Quebec), activated R. leg. bv. viciae 1.00 109 4.94
sludge process B. japonicum 2.32 109 8.67
Quebec city wastewater plant, S. meliloti 3.10 109 7.81
bioWlter process (biodorf) R. leg. bv. viciae 4.30 108 9.21
R. leg. bv. phaseoli 7.70 108 16.00
R. leg. bv. trifolii 3.00 109 9.71
Pulp and paper (chemithermomechanical S. meliloti 3.30 109 6.27
process) wastewater treatment plant, R. leg. bv. viciae 2.80 109 4.19
activated sludge process B. japonicum 1.58 109 8.08
B. elkanii 1.29 109 10.01
Mixed sludge Quebec city wastewater plant: primary S. meliloti 0.85 109 8.25
clariWer sludge and secondary sludge R. leg. bv. viciae n.g. n.g.
obtained from a bioWlter (biodorf) B. japonicum n.g. n.g.
B. elkanii n.g. n.g.
Standard medium (YMB) S. meliloti 4.80 109 5.53
R. leg. bv. viciae 3.80 109 3.92
B. japonicum 4.20 109 7.35
B. elkanii 1.28 109 9.80
n.g.: no growth.
3542 F. Ben Rebah et al. / Bioresource Technology 98 (2007) 35353546

Table 4 bon source represents an important step in the bioprocess.

EVect of suspended solids on S. meliloti growth in sludge from municipal To improve availability of sludge nutrients for rhizobia,
wastewater plants located in Quebec (adapted from Ben Rebah et al.
(2001) and Dufresne (2004))
chemical pre-treatments have been applied to sludge:
acidiWcation with H2SO4 pH 2, alkaline hydrolysis
Suspended solids Maximum cell count Generation
(%) (cfu/mL) time (h)
(185 meqNaOH/l) and peroxide hydrolysis (H2O2 30%)
(Ben Rebah et al., 2001, 2002b; Dufresne, 2004). Growth of
Primary sludge (Black Lake plant)
S. meliloti varied with the sludge origin and the type of pre-
3.2a 0.96 109 9.61
2.6 9.25 109 6.26 treatment; generally, pre-treatments reduced the lag period
1.3 11.1 109 5.34 and the generation time, and enhanced cell yields (Ben
0.65 4.00 109 4.33 Rebah et al., 2001). For example (Table 5), after alkaline
0.325 6.75 109 4.15 treatment of the mixed sludge, the S. meliloti cell count was
Secondary sludge (Black Lake plant) increased from 0.88 109 to 4.1 109 cfu/mL, and the gen-
0.2a 0.99 109 7.34 eration time reduced from 7.87 to 6.83 h. However, for sec-
0.3 0.61 109 7.03 ondary sludge, alkaline treatment reduced cell growth,
0.4 0.66 109 6.90 indicating that this treatment is probably not adequate for
Secondary sludge (Quebec city plant) this type of sludge. Similarly, it was demonstrated by Duf-
0.5 5.55 109 2.30 resne (2004) that the alkaline treatment of the municipal
1.0a 4.15 109 4.00 secondary sludge of Quebec City reduced the lag period of
2.0 5.75 108 ndb
S. meliloti.
a
Suspended solids percentages in the original sludge sample. Other experiments aimed to optimize the alkaline (50
b
nd: not determined.
200 meq/L of NaOH) and acid (pH 2.06.0 obtained with
H2SO4) hydrolysis pre-treatments in sludges having diVer-
probably allowed an optimal oxygen transfer and improved ent solids concentrations (0.3253.2% w/v for primary slud-
cell multiplication, but over-dilution can reduce nutrient ges and 0.20.4% w/v for secondary sludges) (Ben Rebah
content, as shown by approximately 50% less cell in sludge et al., 2001). The best cell yields were obtained in primary
diluted at 0.325% solid concentration (compared to that sludge under alkalinity at 100 meq/L of NaOH with 0.65%
obtained at 1.3% solids concentration). The increase of sus- solids concentration (21 109 cfu/ml) or under acidity at
pended solids concentration from 0.2% to 0.4% in Black pH 2 with 0.325% solid concentrations (13 109 cfu/ml). In
Lake secondary sludge, slightly reduced the maximum cell secondary sludge, the increases in cell yields due to these
count suggesting an inhibitory eVect of factors such as interactions were less important.
heavy metal, Ca and Mg (Ben Rebah et al., 2001; Dufresne, The enhancement of rhizobial yields in pre-treated
2004). Since the maximum solids concentration (0.4% w/v) sludge is probably due to the transformation of the sludge
obtained by concentration of the original sludge (0.2% w/v) complex compounds into available nutrients. It has been
remained very low, it could not have signiWcantly aVected reported by Rajan et al. (1989) that acid hydrolysis solu-
the oxygen transfer during rhizobial growth. bilizes the sludge organic matter. Alkaline and oxidative
treatments of lignocellulosic materials can induce
6.2.3. Optimizing growth conditions in sludge swelling in particulate organics, making the cellular sub-
As stated earlier, inoculants should contain a high cell stances more susceptible to enzymatic attack during sac-
numbers to achieve eYcient nodulation and nitrogen Wxa- chariWcation (Tagaki, 1987; Baccay and Hashimoto,
tion during plant growth. In search of high quality inocu- 1984).
lant, optimizing growth conditions increasing the cell yield Nutrient addition: The eVects of additional sources of
has been achieved by a pre-treatment of sludge or by the nutrients on the growth of S. meliloti were studied in sec-
addition of nutrients. ondary sludge from the Quebec City wastewater plant
Sludge pre-treatments: Sludge contains complex organic (Ben Rebah et al., 2002d). Sludge supplemented with
material, which cannot be easily assimilated by bacteria. diVerent concentrations of yeast extract and glycerol
The transformation of this material into an available car- increased the maximum cell counts (Table 6). The highest

Table 5
EVects of sludge chemical pre-treatments on the growth of S. meliloti in mixed and secondary sludge from municipal wastewater plants (adapted from Ben
Rebah et al. (2001))
Mixed sludge (Quebec city plant) Secondary sludge (Valcartier plant)
Maximum cell count (cfu/mL) Generation time (h) Maximum cell count (cfu/mL) Generation time (h)
9 9
No treatment 0.88 10 7.87 3.60 10 4.96
Acid treatment 2.25 109 7.44 5.80 109 4.93
Alkaline treatment 4.10 109 6.83 0.40 109 10.58
Oxidative treatment 2.14 109 2.14 0.31 109 10.12
 F. Ben Rebah et al. / Bioresource Technology 98 (2007) 35353546 3543

Table 6
EVects of nutrient addition on the growth of S. meliloti in secondary sludge
Nutriment sources Culture vessel Maximum cell count (cfu/mL) Generation time (h) References
Un-supplemented sludge 0.25-L Xask 2.7 109 3.7 Ben Rebah et al. (2002d)
+ Yeast extract (4 g/L) 0.25-L Xask 8.8 109 3.0 Ben Rebah et al. (2002d)
+ Yeast extract (4 g/L) + Glycerol (7.5 g/L) 0.25-L Xask 16.0 109 3.0 Ben Rebah et al. (2002d)
+ Industrial yeast waste (50%) 0.25-L Xask 8.7 109 3.0 Dufresne (2004)
Standard YMB 10-L reactor 3.4 109 2.1 Dufresne (2004)
Un-supplemented sludge 10-L reactor 2.1 109 2.0 Dufresne (2004)
+ Industrial yeast waste (50%) 15-L reactor 6.3 109 2.6 Dufresne (2004)
+ Industrial yeast waste (50%) 150-L reactor 6.9 109 2.6 Dufresne (2004)

increase was approximately sixfold and was obtained and 108 cells/g which is higher than the standard of 107 cells/
with the addition of 4 g/L yeast extract and 7.5 g/L glyc- g used in many countries. Moreover, sludge-based inocu-
erol. Nutrient additions also reduced the generation time. lants can meet the Canadian standard (based on number
This eVect can be explained by the presence in yeast viable cells per seed) because rhizobial counts are higher
extract of amino acids, inorganic nitrogen and growth than the quantity (cells/g) needed to provide the required
factors (iron, calcium, magnesium, strontium, sodium, number of 103 rhizobia per alfalfa seed (e.g. 2 106 per g,
potassium, barium, manganese, copper, lead, aluminium which is an example given in Section 3). Sludge is also non-
and vanadium) at concentrations to satisfy the nutritional toxic, permits growth after incubation, and constitutes an
requirements of rhizobia (Burton, 1979). Brewers spent available and low cost raw material. These characteristics
yeast, an industrial waste recovered from a brewery, has make sludge a suitable carrier for producing solid-based
been used to replace yeast extract in studies with rhizobia inoculants.
growing in sludge because it is a good source of protein,
vitamin and growth factors (Dufresne, 2004). Experi- 6.4. Nodulation capacity and symbiotic eVectiveness of
ments done in Xasks and in 15- and 150-L reactors rhizobia in sludge-based inoculants
showed the beneWcial eVect of spent yeast on S. meliloti
growth (Table 6). It is important to note that rhizobia produced under all
growth conditions in diVerent types of sludges maintained
6.3. Dewatered sludge as a carrier their nodulation capacity (Ben Rebah et al., 2001,
2002b,c,e; Dufresne, 2004). Nodulation indices (nodule size,
Dewatered wastewater sludge has been tested as a car- color and number) were determined on legumes inoculated
rier for S. meliloti by Ben Rebah et al. (2002e). Compared with sludge-grown rhizobia. The nodulation indices of S.
to the control carrier, that is peat inoculated with YMB- meliloti (nodulating alfalfa), R. leguminosarum bv. viciae
grown S. meliloti, dewatered sludge showed optimal poten- (nodulating pea), B. japonicum and B. elkanii (nodulating
tial as a carrier for rhizobia, because it supported rhizobia soya) varied slightly among species and with the origin of
survival at an acceptable level. Sludge can maintain desired sludges, but were generally close to those obtained for
rhizobial populations, a pH around neutral and an accept- YMB-grown rhizobia. Also, the symbiotic eVectiveness of
able water holding capacity after 130 days of storage at sludge-based inoculants (liquid and solid) was compared to
25 C (Table 7). Stored at 4 C, sludge behaved similarly, that of rhizobia grown in the standard medium (YMB)
maintaining populations comparable to those obtained at and/or stored in peat (Ben Rebah et al., 2002c). Alfalfa
25 C. Generally, the viable cell number varied between 107 plants were inoculated with either liquid or solid S. meliloti
inoculants and grown in pots under controlled conditions
in two soils types (clay and sandy soil) previously cultivated
Table 7
with alfalfa and containing indigenous rhizoba (Table 8). In
Survival of S. meliloti and characteristics of sludge-based inoculants (pre-
pared by mixing YMB-grown or sludge-grown S. meliloti with dewatered the sandy soil, solid sludge-based inoculant allowed the
sludge) after 130 days of incubation at 25 C (adapted from Ben Rebah highest alfalfa shoot dry weight while in the clay soil, there
et al. (2002e)) was no signiWcant eVect of inoculation ; however, all types
Inoculants Characteristics of inoculants increased alfalfa nodulation in both soils, as
Carrier Growth Viable cell pH Moisture well as rhizobial populations. For example, for the sandy
media count (cells/g) (%) soil, inoculation increased the nodulation indices of alfalfa
Dewatered sludge Sludge 2.5 107 7.70 39.9 from 4 (control) to 8 and 12 and the number of rhizobia
Dewatered sludge YMB 1.0 108 7.80 42.0 from 103 cells/g to 106 cells/g. The same tendency was
Dewatered sludge + peat Sludge 1.0 108 7.52 35.9 observed for the clay soil. Therefore, inoculation of alfalfa
Dewatered sludge + peat YMB 2.0 108 7.36 39.5 with sludge-based S. meliloti inoculants is as eYcient as
Peat YMB 7.7 108 6.87 36.4 inoculation with standard inoculants.
3544 F. Ben Rebah et al. / Bioresource Technology 98 (2007) 35353546

Table 8 Ben Rebah, F., Tyagi, R.D., Prvost, D., 2001. Acid and alkaline treat-
Growth of alfalfa and populations of S. meliloti in potted soils inoculated ments for enhancing the growth of rhizobia in sludge. Canadian Jour-
with S. meliloti produced in sludge and by standard procedure (adapted nal of Microbiology 47, 467474.
from Ben Rebah et al. (2002c)) Ben Rebah, F., Filali-Meknassi, Y., Yezza, A., Tyagi, R.D., Surampalli,
R.Y., 2002a. Wastewater treatment as an approach for the recovery of
NIa/pot Shoot dry MPNc
value added products. In: IAWQ. Water and Wastewater: Prospectives
weightb in soils
of Developing Countries, Proceedings of the International Conference,
Sandy soil WAPDEC, pp. 965972.
Non-inoculated 4 10.70 3.25 103 Ben Rebah, F., Tyagi, R.D., Prvost, D., Surampalli, R.Y., 2002b. Waste-
Liquid inoculant (YMBd) 8 10.39 2.27 106 water sludge as a new medium for rhizobial growth. Water Quality
Liquid inoculant (sludge-based) 8 10.90 5.06 106 Research Journal of Canada 37, 353370.
Solid inoculant (standard-YMB-peat) 8 10.18 nde Ben Rebah, F., Tyagi, R.D., Prvost, D., 2002c. Nodulation and yield of
Solid inoculant (sludge) 12 13.07f nd alfalfa grown in sludge amended soils and inoculated with rhizobia
Clay soil produced in sludge. Journal of Environmental Quality 31, 13391348.
Non-inoculated 6 15.83 4.83 10c Ben Rebah, F., Tyagi, R.D., Prvost, D., 2002d. Production of S. meliloti
Liquid inoculant (YMB) 8 15.03 2.06 107 using wastewater sludge as a raw material: eVect of nutrient addition
Liquid inoculant (sludge-based) 8 15.00 1.62 107 and pH control. Environmental Technology 23, 623629.
Solid inoculant (standard-YMB-peat) 12 16.65 nd Ben Rebah, F., Tyagi, R.D., Prvost, D., 2002e. Wastewater sludge as a
Solid inoculant (sludge) 12 15.94 nd substrate for growth and carrier for rhizobia. Bioresource Technology
83, 145151.
a
Nodulation indices at the end of experiment. Bissonnette, N., Lalande, R., 1988. High survivability of cheese whey-
b
Total of three cuts (g/pot). grown Rhizobium meliloti cells upon exposure to physical stress.
c
Most probable number of rhizobial cells at the end of experiment Applied and Environmental Microbiology 54, 183187.
(cells/g soil). Bissonnette, N., Lalande, R., Bordeleau, L.M., 1986. Large-scale produc-
d
YMB: yeast mannitol broth. tion of Rhizobium meliloti on whey. Applied and Environmental
e
nd: not determined. Microbiology 52, 838841.
f
SigniWcantly diVerent (among sandy soil treatments). Boiardi, J.L., Ertola, R.J., 1985. Rhizobium biomass production in batch
and continuous culture with a malt-sprouts medium. World Journal of
Microbiology and Biotechnology 1, 163172.
7. Conclusions Brockwell, J., Bottomley, P.J., 1995. Recent advances in inoculant technol-
ogy and prospects for the future. Soil Biology and Biochemistry 27,
This review has considered the use of waste in the produc- 683687.
tion of liquid and solid rhizobial inoculants. Many agro- Burton, J.C., 1967. Rhizobium culture and use. In: Peppler, H.J. (Ed.),
Microbial Technology. Reinhold Publishing Corp., New York, pp. 1
industrial wastes showed good potential to replace the stan- 33.
dard medium and the commonly used peat in inoculant pro- Burton, J.C., 1979. Rhizobium species. In: Peppler, H.J., Perlman, D. (Eds.),
duction. Although availability, transport, cost and treatment Microbial Technology: Microbial Processes, second ed., vol. 1. Aca-
of these wastes may limit inoculant production, their use still demic, New York, pp. 2958.
oVers a valuable alternative for recycling wastes that sup- Campbell, H.W., Martinoli, D.K., 1991. Canadas oil-from-sludge technol-
ogy. Water Environment and Technology 3, 6466.
ports sustainable agriculture. The use of industrial and Catroux, G., 1991. Inoculant quality standards and controls in France. In:
municipal wastewater sludge remains a challenging but new Thompson,V.A. (Ed.), Expert Consultation on Legume Inoculant Pro-
option for sludge disposal and recycling, and may reduce the duction and Quality Control. FAO, Roma 19-21 March 1991, Italy,
production cost of inoculants. Compared to other wastes, pp. 113120.
sludge is an abundant raw material for inoculum prepara- Catroux, G., Hartman, A., Revellin, C., 2001. Trends in rhizobial inoculant
production and use. Plant and Soil 230, 2130.
tion. Sludge can be used without any treatment process Cerbulis, J., Woychik, H., Wondolowski, M.V., 1982. Composition of com-
because of its organic composition (C, N, P, solids) and its mercial whey. Journal of Agricultural and Food Chemistry 20, 1057
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