Agro-industrial waste materials and wastewater sludge for rhizobial - 2007

Bioresource Technology 98 (2007) 3535–3546

Agro-industrial waste materials and wastewater sludge for rhizobialinoculant production: A review

F. Ben Rebah a, D. Prévost b,¤, A. Yezza c, R.D. Tyagi c

a INSTM-Sfax B.P. 1035, Sfax 3018, Tunisiab Agriculture et Agroalimentaire Canada, Sainte-Foy, QC, Canada G1V 2J3

c INRS-Eau, Terre et Environnement (INRS-ETE), 490 rue de la Couronne, Québec, QC, Canada G1K 9A9

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

Abstract

Inoculating legumes with commercial rhizobial inoculants is a common agriculture practice. Generally, inoculants are sold in liquid orin solid forms (mixed with carrier). The production of inoculants involves a step in which a high number of cells are produced, followedby 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, maltsprouts) 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 usuallycontains nutrient elements at concentrations suYcient to sustain rhizobial growth and heavy metals are usually below the recommendedlevel. In some cases, growth conditions can be optimized by a sludge pre-treatment or by the addition of nutrients. Inoculants produced inwastewater sludge are eYcient for nodulation and nitrogen Wxation with legumes as compared to standard inoculants. This new approachdescribed 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

The rhizobium-legume symbiosis plays an importantrole in agriculture, because it oVers the ability to convertatmospheric molecular nitrogen into forms useable by theplant, a process called biological nitrogen Wxation. Most ofthe research to optimize symbiotic nitrogen Wxation and toincrease the use of legumes in crops systems has been inpart stimulated by increasing fertilizer prices and by envi-ronmental concerns. One beneWt of increased knowledge ofsymbiotic nitrogen Wxation is the inoculation of legumes byselected rhizobia, a practice that allows producers to intro-duce rhizobia into the soil–plant ecosystem. Inoculants are

* Corresponding author. Tel.: +1 418 657 7980; fax: +1 418 648 2402.E-mail address: [email protected] (D. Prévost).

0960-8524/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2006.11.066

commercial formulations containing rhizobia that can beapplied to the seed or the soil during planting (reviewed byBrockwell and Bottomley, 1995). It was estimated thatapproximately 2000 tons of inoculants are producedworldwide annually, a quantity suYcient to inoculate 20million ha of legumes. The largest single producers are inthe USA with an annual production of 1000 tons (Single-ton et al., 1997). Inoculants are usually commercialized assolid inoculants, in powder from peat or in granular form,or as liquid inoculants, in broth formulations (Stephensand Rask, 2000). Alternative formulations such as oilbased products or concentrates, lyophilized cultures or rhi-zobial cells entrapped in beads have been investigated. Themethod of inoculation depends on the inoculant type: pow-dered and liquid inoculants are generally applied to theseeds, while granular inoculants are applied to the soil, inthe furrow.

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3536 F. Ben Rebah et al. / Bioresource Technology 98 (2007) 3535–3546

Solid inoculants are prepared by adding fermenter-grown broth, containing a large population of rhizobium,to a powdered carrier followed by a period of maturationto ensure multiplication or adaptation of rhizobial cells(Roughley and Pulsford, 1982). Thus, the production of alarge quantity (greater than 109 cells per mL) of rhizobia inliquid medium constitutes the Wrst step in the productionof powdered legume inoculants. The economies of this pro-cess are governed by the cost and availability of a suitablecarbon source (Bissonnette et al., 1986). The standardmedium, including mannitol as carbon source and yeastextract as a source of nitrogen, growth factors and mineralsalts, has been used for laboratory scale production, but itsindustrial use is limited due to high cost. Among carriersthat can sustain high levels of rhizobia, peat is the mostwidely used (Burton, 1967; Peterson and Loynachan,1981), but is not universally available (Tilak and SubbaRao, 1978; Corby, 1976). Alternatively, diVerent materialssuch as industry by-products, organic wastes, mineral soils,plant by-products, coal, perlite, and agro-industrial wasteshave 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 easilyavailable in a speciWc region. In this review we will presentthe potential of diVerent products and wastes tested asgrowth substrates and carriers for rhizobia. Emphasis willbe given to wastewater sludges which have been extensivelycharacterized and have proven to be very good material forinoculants production.

2. Need for inoculation

Biological nitrogen Wxation by rhizobia representsapproximately 70–80% of the total nitrogen accumulatedby legume plants. Estimates of nitrogen Wxation in legumecrops are 105 and 200 kg N ha¡1 yr¡1 for clover, 169 kgN ha¡1 yr¡1 for lupine, 208 kg N ha¡1 yr¡1 for alfalfa and234 kg N ha¡1 yr¡1 for soybean (Brockwell and Bottomley,1995). This process has the advantage of reducing thedependency of agriculture on nitrogen fertilizers, and it iseconomical, using energy from photosynthesis. Fixed nitro-gen is beneWcial to soil fertility and reduces the environ-mental pollution associated with the use of nitrogenfertilizers.

Several soil and environmental stresses aVect the perfor-mance of the legume-rhizobium symbiosis and may limitnodulation and nitrogen Wxation. Rhizobial populations,the host plant species, agronomic practices and climaticconditions are among the important factors. When a newcrop 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). Sincerhizobia should be in suYcient numbers to ensure optimalnodulation and eYcient nitrogen Wxation, inoculation oflegumes 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 acentury, and they have contributed to increased crop pro-ductivity. A good inoculant must be prepared with a strainof rhizobium selected for high N Wxation eYciency andcompetitive ability for nodulation. The strain must survivein the inoculant formulation, maintain its properties duringstorage, and be tolerant to stress factors such as acidity,desiccation, high temperature and chemical pesticides. Themost 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 notdetrimental to rhizobia or pathogen to plants and humans(Lupwayi et al., 2000).

Many countries, including Australia, the Netherlands,Rwanda, and Thailand have regulated minimum standardsvarying from 5£107 to 1£109 rhizobium cells/g of inoculantproduct (Lupwayi et al., 2000). In Canada, the standardsstipulate a minimum number of rhizobia delivered per seed:103 rhizobia per small seed (e.g. clover and alfalfa), 104 permedium seed (e.g. mungbean, pigeonpea, sainfoin) and 105

per large seed (e.g. soybean, pea and bean). Thus, the numberof rhizobia required in an inoculant product depends on thehost legume species (seed size) and on the rate of applicationof the product suggested by the manufacturer. For example,for inoculant products sold in 1997 in Canada, the minimalnumber of rhizobia required to meet the standards variedfrom 2£106 to 1.3£109 rhizobium cells per g or mL product(CFIA, 1997). France determined a minimum standard of106 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 andthe 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 characteristicfor farmers and manufacturers. Inoculant manufacturersprefer products with a shelf-life of 1–1.5 years under ware-house conditions in order to be able to sell them over twocropping seasons (Catroux et al., 2001). Many inoculantsproduced with local alternative carriers can maintain accept-able numbers of rhizobia for 3–12 months (see Section 5,production of legume inoculants), which is suYcient if theyare used shortly after their production.

4. Nutrient requirements of rhizobia

Based on growth rate, nutritional requirement andenzymes, rhizobia can be traditionally divided in twogroups: the fast-growing rhizobia, placed in the genus Rhi-zobium and the slow-growing rhizobia represented by thegenus Bradyrhizobium. More recently, phenotypic and geno-typic characterization of rhizobia isolated from an increas-ing number of plant species has led to the reclassiWcationand new designation of some species and to the descriptionof 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) 3535–3546 3537

cies commonly used in legume inoculants will be designatedaccording 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) foralfalfa; Rhizobium leguminosarum bv. viciae (R. leguminosa-rum) for peas, lens and vetches; bv. phaseoli (R. phaseoli) forbeans; bv. trifolii (R. trifolii) for clover; Rhizobium tropicifor 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 growthrequirements. This knowledge is useful in selecting organicwastes 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 2–4 mm diameter, after 3–5 days of incubation onyeast mannitol agar (YMA) (Jordan, 1984). Slow-growingrhizobia (Bradyrhizobium) are alkali-producers; theyexhibit a doubling time of 6–8 h and form colonies notexceeding 1 mm in diameter after 5–7 days of incubation onYM media (Jordan, 1984).

Fast- and slow-growing rhizobia also diVer in theircapacity to utilize carbohydrates. Fast-growing bacteriacan grow on a large variety of carbon substrates, such ashexoses, pentoses, disaccharides, trisaccharides and organicacids (Allen and Allen, 1950). Slow-growing rhizobia havea more restricted range of carbon sources, they grow in thepresence of pentoses, and they can also utilize many aro-matic substrates (Parke and Ornston, 1984).

All rhizobial species require nitrogen sources, vitaminsand trace minerals for growth. Some fast-growing rhizobiacan utilize nitrate, ammonia and amino acids as their solenitrogen source (Quispel, 1974). Optimal growth can beachieved by adding low weight amino acids, but the balanceof amino acids is important (Burton, 1979). However, rhi-zobia grown on media enriched with glycine, alanine andcertain D-forms of amino acids loose the capacity of nitro-gen Wxation (Burton, 1979). Vitamin requirements varyamong rhizobial species; R. leguminosarum (bv. trifolii andbv. 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 satisWesneeds of rhizobia.

Rhizobia also require mineral salts for growth. Yeastextract contains signiWcant concentrations of salts (K, Ca,Mg, and others) (Burton, 1979). Calcium deWciency aVectsrhizobial growth, and low concentrations (0.1 ppm) produceabnormal cells (Vincent, 1962). InsuYcient quantities of Mgcause an abnormal cell multiplication (Norris, 1959). Certaintrace minerals such as cobalt, zinc, manganese and iron are

required at low concentrations for rhizobial growth (Loweand Evans, 1962; Wilson and Reisenauer, 1970a,b).

Yeast extract can be used as a sole carbon and nitrogensource 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 concentrationfor rhizobial growth (Burton, 1979). The addition of yeastextract to growth media improves the eYciency of rhizobialgrowth (Meade et al., 1985; Bissonnette and Lalande, 1988;Ben Rebah et al., 2002d), but high concentrations are noteconomical for large industrial production. Concentrationshigher than 0.35% are not beneWcial because they producedistorsed cells and decrease the viability of some rhizobialstrains (Skinner et al., 1977).

5. Production of legume inoculants

5.1. Quantity production of rhizobia in liquid cultures

5.1.1. Standard mediumThe 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 introductionof other carbon sources: sucrose is the most commonlyused carbon source, and mannitol and glycerol aresometimes 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-productsNumerous 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 culturemedia, as shown by the high quantity of rhizobial cells pro-duced (Table 1).

Cheese whey: The by-product of the cheese industry wasused 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 70–75% of the total solids while nitro-gen represents 1.82–2.4%. The lactose uptake system of S.meliloti is inducible, and its catabolism involves cleavage tomonosaccharides 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 andgave a cell population similar to that obtained in YMB. Inwhey media, stationary growth was reached after 48h, andthe maximum cell number of 6.3£109 cfu/mL was obtainedwithin 72 h. Whey supplemented with yeast extract (1g/L)and phosphate (0.5g/L) increased the cell number to1£1010 cfu/mL after 48 h (Bissonnette et al., 1986). The same


authors demonstrated that whey protects rhizobial cells fromfreezing and thawing damage, since S. meliloti grown inwhey survived freezing at ¡18 °C better than cells grown onmannitol or sucrose (Bissonnette and Lalande, 1988). More-over, rhizobia produced in whey and stored in peat survivedbetter at various temperatures of storage as shown by 100%survival after 23 weeks at – 4°C. Therefore, the by-productof the dairy industry oVers an interesting potential as growthmedia and as protectant for industrial inoculants.

Malt sprouts: The by-product of the malt industry (maltsprouts) 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 at100 °C for 30 min. After centrifugation, a liquid wasobtained that contained 3.75% w/v of solids, 0.18% w/v ofnitrogen 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 containingdiVerent concentrations of malt sprouts extract. The maltsprouts extract at concentrations varying from 0 to 80% v/vof the growth medium were used to replace yeast extract inthe medium containing: glycerol, yeast extract, peptone,K2HPO4, KH2PO4, MgSO4 ·7H2O, FeCl3 ·6H2O, NaCl,MnSO4 and biotin. In batch culture, all strains grew well inthe malt sprouts based medium and reached a concentrationhigher than 5£109 cfu/mL. DiVerences between slow andfast-growers were observed: cell yields of the fast-growingrhizobia increased with increasing concentration of the maltsprouts extract, while a concentration of 40% v/v inhibitedthe growth of the slow-growing rhizobia (B. japonicum).

Industrial-grade yeast extract: With the aim to producecell concentrates of R. leguminosarum bv. viciae, Meadeet al. (1985) compared standard YMB with a medium usingindustrial-grade yeast extract as the carbon and nitrogensource under industrial-scale conditions. The growth yieldin yeast extract (5 g/L) medium was 2.4£109 and8.1£109 cfu/mL for medium containing 0.6 and 0.9 g/L ofphosphate buVer respectively, and these values were similarto that of 3£ 109 cfu/mL obtained in standard YMB

medium. Yeast extract was advantageous for cell recoverywith a continuous centrifugation system since the recoverywas 63% and 70% in yeast extract medium in comparisonto only 0.6% in standard YMB medium. The better recov-ery in yeast extract medium was attributed to the absenceof polysaccharides which, in the YMB medium, trap cellwithin the viscous supernatant.

Pea husk, molasses and water hyacinth: Agriculturewastes were used to formulate a non-synthetic medium(Gulati, 1979) that contained pea husk, molasses, waterhyacinth, 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 partiallyreplace the yeast extract normally present in standardYMB. Many strains of R. leguminosarum bv. trifolii and B.japonicum tested in Xask and in fermentors for large-scaleproduction showed equal growth in standard and in formu-lated non-synthetic media. The suitability of the formulatedmedium can be attributed to the presence of requiredamino acids such as tryptophan, glutamic acid and carbonsources such as glucose, arabinose and cellobiose. Thelarge-scale production (25 and 135 L fermentors) was ade-quate for commercial use.

Jaggery: Jaggery is a coarse brown Indian sugar madefrom palm-sap that has been tested as sole source of carbonfor three strains of B. japonicum and three strains of Rhizo-bium sp. (Jain et al., 2000). In general, the number of cellsobtained in jaggery was similar to that obtained in manni-tol or glycerol media, but a few strains grew faster and gavea higher number of cells in jaggery.

5.2. Alternative carriers for production of solid inoculants

Most inoculants on the market are prepared in powderedorganic carriers such as peat by adding large populations ofrhizobia 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

Table 1Production of biomass from diVerent species of rhizobia grown in agricultural and industrial by-products

a Growth time in parentheses.

Wastes Rhizobial strains Type of culture vessel Max cell count (cells/mL) References

Whey S. meliloti 5-L fermentor (48 h)a 4.7 £ 109 Bissonnette et al. (1986)

Malt sprouts S. meliloti 1-L Xask >6 £ 109 Boiardi and Ertola (1985)S. meliloti 5-L fermentor >5 £ 109

R. leguminosarum bv. phaseoli 1-L Xask >3 £109

R. leguminosarum bv. viciae 1-L Xask >5 £ 109

B. japonicum 1-L Xask >5 £ 109

Grade yeast extract R. leguminosarum bv. viciae 200-L fermentor (40 h) 2.4 £ 109 to 8.1 £ 109 Meade et al. (1985)

Pea husk, molassesand water hyacinth

R. leguminosarum bv. trifolii 25-L fermentor (72 h) 2.80£ 1010 Gulati (1979)R. leguminosarum bv. trifolii 135-L fermentor (72 h) 1.75£ 1010

B. japonicum 25-L fermentor (96 h) 2.20£ 1010

B. japonicum 135-L fermentor (84 h) 1.75£ 1010

B. japonicum and R. sp. (144 h) 9.86£ 109 Jain et al. (2000)


(Lupwayi et al., 2000). Therefore, a carrier should be high inorganic matter with optimum nitrogen content, a pHaround neutral and a high water holding capacity. It shouldbe non-toxic, permit growth after incubation, be availableall year-round and be inexpensive. Peat is the most widelyused carrier, and it can sustain high numbers of rhizobia(greater than 108 cells/g) during storage at a temperaturerange from 3 to 28 °C (Burton, 1967; Van Schreven, 1970).Granular preparations using peat prill or mineral granulesare also suitable for inoculation of diVerent farming systems(Stephens and Rask, 2000). Since peat is not available in anumber of countries, particularly in regions without naturalsdeposits 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 availablewaste materials. Viability of rhizobia is the most importantfactor, and data for some carriers are listed in Table 2.

It is rather diYcult to compare results from diVerentexperiments using diVerent storage conditions. As seen inTable 2, the storage temperature was either cold (4 °C or10 °C) or ambient (20, 25 or 30 °C), and the length of stor-age varied from 24 days to 12 months. Despite of thesevarying 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 termstorage (35 weeks) with populations higher than 108 cells/g,while only 106 cells/g were observed in mineral soils after 15weeks (Chao and Alexander, 1984). All plant materials sus-tained populations varying from 107 to 108 cells/g exceptcellulose powder which only sustained 106 cells/g after 24days (Pugashetti et al., 1971). In general, inert materialshowed a better potential than plant material. In polyvinyl-pyrrolidone (PVP), populations were higher than 109 cells/g(Denardin and Freire, 2000) and 108–109 cells/g survived invermiculite (Graham-Weiss et al., 1987), perlite (Daza et al.,2000) and bentonite (Choi et al., 1998). Rhizobia entrappedin beads of alginate (Jung et al., 1982) seem to survive bet-ter than those entrapped in polyacrylamide (Dommergueset al., 1979). Recently, local agro-industrial wastes such asoxalic acid production waste (Ajay et al., 1996) and Xy-ash(Ghosh et al., 1996) have been successfully used as carriersfor the production of rhizobia. The alternative carriers havethe 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 canbe eliminated by physical, chemical and or biological treat-

ment processes. These treatments generate sludge, which isconsidered recyclable waste. Sludge is a complex suspen-sion containing water (between 95% and 98%), organic andinorganic matter and a large variety of microorganisms.Sludge is classiWed into primary, secondary or digestedsludge depending on the nature of the treatment process,the aZuent origin (municipal or industrial) and the rate ofpollutant elimination (Roques, 1979).

The province of Quebec (Canada) generated 218,000tons of sludge (dry weight) in 2002 (Hébert, 2004). TheUSA 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 problemand 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 andMartinoli, 1991) and building materials (Tay and Show,1992) are some of the approaches used for sludge disposaland recycling. Recently, emphasis has been placed on thebioconversion of sludge into value-added products such asbiopesticides (Lachhab et al., 2001), enzymes (Tyagi et al.,2002) and bioplastics (Ben Rebah et al., 2002a). The nextsections of this review will present evidences showing thatwastewater sludge also constitutes an eVective alternativeto produce rhizobium for legume inoculants.

6.2. Sludge as a growth media

6.2.1. EVects of sludge composition on rhizobial growthGenerally, the organic and mineral composition of

sludge varies depending on the origin and the treatmentprocess. However, sludges generated by municipal andindustrial wastewater treatment processes (Canada) usedby 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 ofwastewater sludge as a sole raw material are the presence oftoxic heavy metals and pathogens. For pathogen removal,the sludge is sterilized before rhizobial inoculation. As faras 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 beclassiWed as Class A, B or C according to the Environmen-tal Protection Agency (EPA, 1993). Class A sludge can besafely used in production of bacterial inoculant (Yezzaet al., 2005). In all types of sludges studied for rhizobialinoculant 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 theEuropean Economic Community. It is however worthy tomention that heavy metals can inhibit the growth of rhizo-bia, depending of their concentration (Wilson and Reis-enauer, 1970a,b). Moreover, long exposure to metalsderived from past applications of sludges in soils may aVectsurvival of eVective rhizobia, and consequently nitrogenWxation (McGrath et al., 1988; Giller et al., 1989). Zn and


Cd are the most toxic metals on survival of R. leguminosa-rum bv. trifolii, but toxicity was observed only after eightmonths at concentrations of 1.3 and 2.4 times the UnitedKingdom limits (Chaudri et al., 1992, 1993). Therefore, it is

unlikely that rhizobia grown in sludges will be severelyaVected by heavy metals because sludge inhibitory factorsare generally complexed by the organic matter. Moreover,the low inoculation rate of legume seeds (2–3 mL or 3–6 g

Table 2Alternative carriers used for solid-based-rhizobial inoculant

Carrier tested Rhizobial species Viability cells per g or mL or per seed References

Soil-based carriersMineral soils R. leguminosarum bv. phaseoli 106 at 4 °C and 105 at 25 °C, 105 d

Viability on coated seeds: mineral soil > peatChao andAlexander (1984)

Coal (8 types) R. leguminosarum bv. phaseoli 107 to 1010 at 20– 22 °C, 4 wksViability on coated seeds: coal 104 < peat 105

Crawford andBerryhill (1983)

Volcanic pumice(nutrient supplemented)

Bradyrhizobium sp. (Lupinus) 108–109 at 22 °C, 35 wks Einarsson et al. (1993)

Inert material-based carriersVermiculite

(nutrient supplemented)B. japonicum, S. meliloti andR. leguminosarum bv. phaseoli

108–109 at ambient T, 4 wks (by directfermentation of initial inoculum)

Graham-Weiss et al. (1987)

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)

6.1 £ 107 at 30 °C, 75 d (Peat: 1 £ 106)Dommergues et al. (1979).

(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(PVP), xanthan, Jatai gum (carob)

B. japonicum >109 at ambient T, 8 mo Denardin andFreire (2000)

Perlite R. leguminosarum bv. phaseoli,R. tropici and B. japonicum

– Similar to peat at 4°C, 6mo– Better than peat at 28°C, 6mo– On coated seeds: similar to peat

with sucrose as adhesive, but notwith gum arabic

Daza et al. (2000)

Agro-industrial wastes-based-carriersSoybean meal and coir dust

(added to soil)R. leguminosarum bv. trifolii Soil + soybean meal: 10 £ 107;

Soil + coir dust: 16 £ 106; Soil: 7 £ 107Iswaran (1972)

Rice husk R.leguminosarum bv. trifolii and bv.viciae, S. meliloti and B. japonicum

1.1–51.2 £ 107 at 28–31 °C, 12 wks Khatri et al. (1973)

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

B. japonicum4.5–7.2 £ 109 at 26 °C, 8 wks Iswaran et al. (1973)

Sawdust (composted) (1) B. japonicum (1) 13.9 £ 108 at 6–9 °C, 9 mo Kostov and Lynch (1998)(2) S. meliloti (2) 15 £ 109 at 6–9 °C, 9 mo(3) M. loti (3) 7 £ 109 at 6–9 °C, 9 mo

Teak leaf meal, compost, charcoal,Indian peat, lignite and mixtures

B. japonicum 1.7–8.5 £ 109 at 30° C, 15 d (comparableto peat: 8.2 £ 109)

Chakarapani andTilak (1974)

(1) Sugarcane bagasse, sawdust Rhizobium sp. (Sesbania) (1) 105–106 at 30 °C, 90 d Muniruzzaman andKhan (1992)(2) Filter mud (2) 107 at 30 °C, 90 d

Sugarcane press mud(alone or mixed)

B. japonicum 3 £ 106 at 30°C, 45 d Singh et al. (1992)

Filter mud R. leguminosarum bv. trifolii andR. sp. (Cowpea)

108–109 at ambiant T, 12 wks (air dried,unsterile and autoclaved)

Philpotts (1976)

Sugar mills B. japonicum 2.6–7.2 £ 108 at ambient T, 3 mo Marafu et al. (1995)

Cotton stalks, groundnut shells,jowar stubbles (degraded)

B. japonicum 1.2 £ 107–2.2 £ 109 at ambient T, 3 mo Potdukhe andSomani (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,Acacia, Sesbenia, Dalbergia)

1.4–2.4 £ 109 at ambient T, 3 mo1.6–10.2 £ 107 at ambient T, 12 mo

Ghosh et al. (1996)

Oxalic acid industrialwaste (OIW)

B. japonicum 109 at room T, 90 d Ajay et al. (1996)


of inoculant per kg of seeds) will introduce only very smallquantities of heavy metals in soils.

Ben Rebah et al. (2002b) demonstrated for the Wrst timethat sludge generated by industrial and municipal wastewa-ter treatment processes sustains growth of various strainsof 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 obtainedwith standard YMB. In a few sludges (e.g. Valcartier pri-mary sludge and the mixed sludge Quebec City), growth ofR. leguminosarum bv. viciae was inhibited and growth of S.meliloti was reduced to 108 cfu/ml. Population levels ofslow-growing rhizobia (Bradyrhizobium) were often lower(108–109 cfu/ml) than those of fast-growing species andlower than those obtained in standard YMB medium. BothB. japonicum and B. elkanii could not grow in the Valcartierprimary sludge nor in the mixed sludge, indicating a lack ofavailable nutrient requirements or a toxicity eVect.

Therefore, rhizobial growth depends on the species andon the sludge characteristics that aVect the growth time andthe cell yield. The Ca/Mg ratio seems to have more impacton 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 rangedbetween 10 and 1/30, while S. meliloti grew better underlower ratio conditions (Steinborn and Roughley, 1975). Instudies with wastewater sludges, the two most inhibitorysludges (Table 3) had a low (2.13, Valcartier primarysludge) and a high (6.7, Quebec city mixed sludge) Ca/Mgratio, 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 suspendedsolids in sludge aVected the growth of S. meliloti in primaryand 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 thesame sludge diluted to 1.3% solid concentration. Reducingthe solids concentrations by dilution of the original sludge

Table 3Growth 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))

n.g.: no growth.

Sludge type Origin and process Strains tested Maximum cell count(cfu/mL)

Generationtime (h)

Primary sludge Municipal wastewater plant,Black Lake (Quebec)

S. meliloti 1.12 £ 109 10.22R. leg. bv. viciae 3.10 £ 109 7.50B. japonicum 0.67 £ 109 8.82B. elkanii 1.20 £ 109 11.37

Municipal wastewater plant,Valcartier (Quebec)

S. meliloti 0.70 £ 109 11R. leg. bv. viciae n.g. n.g.B. japonicum n.g. n.g.B. elkanii n.g. n.g.

Secondary sludge Municipal wastewater plant,Black Lake (Quebec), sequentialbatch reactor


Municipal wastewater plant,Valcartier (Quebec), activatedsludge process

S. meliloti 2.45 £ 109 6.52R. leg. bv. viciae 1.00 £ 109 4.94B. japonicum 2.32 £ 109 8.67

Quebec city wastewater plant,bioWlter process (biodorf)

S. meliloti 3.10 £ 109 7.81R. leg. bv. viciae 4.30 £ 108 9.21R. leg. bv. phaseoli 7.70 £ 108 16.00R. leg. bv. trifolii 3.00 £ 109 9.71

Pulp and paper (chemithermomechanicalprocess) wastewater treatment plant,activated sludge process


Mixed sludge Quebec city wastewater plant: primaryclariWer sludge and secondary sludgeobtained from a bioWlter (biodorf)

S. meliloti 0.85£ 109 8.25R. leg. bv. viciae n.g. n.g.B. japonicum n.g. n.g.B. elkanii n.g. n.g.

Standard medium (YMB) S. meliloti 4.80 £ 109 5.53R. leg. bv. viciae 3.80 £ 109 3.92B. japonicum 4.20 £ 109 7.35B. elkanii 1.28 £ 109 9.80


probably allowed an optimal oxygen transfer and improvedcell multiplication, but over-dilution can reduce nutrientcontent, as shown by approximately 50% less cell in sludgediluted at 0.325% solid concentration (compared to thatobtained at 1.3% solids concentration). The increase of sus-pended solids concentration from 0.2% to 0.4% in BlackLake secondary sludge, slightly reduced the maximum cellcount suggesting an inhibitory eVect of factors such asheavy metal, Ca and Mg (Ben Rebah et al., 2001; Dufresne,2004). Since the maximum solids concentration (0.4% w/v)obtained by concentration of the original sludge (0.2% w/v)remained very low, it could not have signiWcantly aVectedthe oxygen transfer during rhizobial growth.

6.2.3. Optimizing growth conditions in sludgeAs stated earlier, inoculants should contain a high cell

numbers to achieve eYcient nodulation and nitrogen Wxa-tion during plant growth. In search of high quality inocu-lant, optimizing growth conditions increasing the cell yieldhas been achieved by a pre-treatment of sludge or by theaddition of nutrients.

Sludge pre-treatments: Sludge contains complex organicmaterial, which cannot be easily assimilated by bacteria.The transformation of this material into an available car-

Table 4EVect of suspended solids on S. meliloti growth in sludge from municipalwastewater plants located in Quebec (adapted from Ben Rebah et al.(2001) and Dufresne (2004))

a Suspended solids percentages in the original sludge sample.b nd: not determined.

Suspended solids(%)

Maximum cell count(cfu/mL)

Generationtime (h)

Primary sludge (Black Lake plant)3.2a 0.96£ 109 9.612.6 9.25£ 109 6.261.3 11.1£ 109 5.340.65 4.00£ 109 4.330.325 6.75£ 109 4.15

Secondary sludge (Black Lake plant)0.2a 0.99£ 109 7.340.3 0.61£ 109 7.030.4 0.66£ 109 6.90

Secondary sludge (Quebec city plant)0.5 5.55£ 109 2.301.0a 4.15£ 109 4.002.0 5.75£ 108 ndb

bon source represents an important step in the bioprocess.To improve availability of sludge nutrients for rhizobia,chemical pre-treatments have been applied to sludge:acidiWcation with H2SO4 pH 2, alkaline hydrolysis(185 meqNaOH/l) and peroxide hydrolysis (H2O2 30%)(Ben Rebah et al., 2001, 2002b; Dufresne, 2004). Growth ofS. meliloti varied with the sludge origin and the type of pre-treatment; generally, pre-treatments reduced the lag periodand the generation time, and enhanced cell yields (BenRebah et al., 2001). For example (Table 5), after alkalinetreatment of the mixed sludge, the S. meliloti cell count wasincreased from 0.88£ 109 to 4.1£ 109 cfu/mL, and the gen-eration time reduced from 7.87 to 6.83 h. However, for sec-ondary sludge, alkaline treatment reduced cell growth,indicating that this treatment is probably not adequate forthis type of sludge. Similarly, it was demonstrated by Duf-resne (2004) that the alkaline treatment of the municipalsecondary sludge of Quebec City reduced the lag period ofS. meliloti.

Other experiments aimed to optimize the alkaline (50–200 meq/L of NaOH) and acid (pH 2.0–6.0 obtained withH2SO4) hydrolysis pre-treatments in sludges having diVer-ent solids concentrations (0.325–3.2% w/v for primary slud-ges and 0.2–0.4% w/v for secondary sludges) (Ben Rebahet al., 2001). The best cell yields were obtained in primarysludge under alkalinity at 100 meq/L of NaOH with 0.65%solids concentration (21£ 109 cfu/ml) or under acidity atpH 2 with 0.325% solid concentrations (13£ 109 cfu/ml). Insecondary sludge, the increases in cell yields due to theseinteractions were less important.

The enhancement of rhizobial yields in pre-treatedsludge is probably due to the transformation of the sludgecomplex compounds into available nutrients. It has beenreported by Rajan et al. (1989) that acid hydrolysis solu-bilizes the sludge organic matter. Alkaline and oxidativetreatments of lignocellulosic materials can induceswelling in particulate organics, making the cellular sub-stances more susceptible to enzymatic attack during sac-chariWcation (Tagaki, 1987; Baccay and Hashimoto,1984).

Nutrient addition: The eVects of additional sources ofnutrients on the growth of S. meliloti were studied in sec-ondary sludge from the Quebec City wastewater plant(Ben Rebah et al., 2002d). Sludge supplemented withdiVerent concentrations of yeast extract and glycerolincreased the maximum cell counts (Table 6). The highest

Table 5EVects of sludge chemical pre-treatments on the growth of S. meliloti in mixed and secondary sludge from municipal wastewater plants (adapted from BenRebah 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)

No treatment 0.88 £ 109 7.87 3.60 £ 109 4.96Acid treatment 2.25 £ 109 7.44 5.80 £ 109 4.93Alkaline treatment 4.10 £ 109 6.83 0.40 £ 109 10.58Oxidative treatment 2.14 £ 109 2.14 0.31 £ 109 10.12


increase was approximately sixfold and was obtainedwith the addition of 4 g/L yeast extract and 7.5 g/L glyc-erol. Nutrient additions also reduced the generation time.This eVect can be explained by the presence in yeastextract of amino acids, inorganic nitrogen and growthfactors (iron, calcium, magnesium, strontium, sodium,potassium, barium, manganese, copper, lead, aluminiumand vanadium) at concentrations to satisfy the nutritionalrequirements of rhizobia (Burton, 1979). Brewer’s spentyeast, an industrial waste recovered from a brewery, hasbeen used to replace yeast extract in studies with rhizobiagrowing in sludge because it is a good source of protein,vitamin and growth factors (Dufresne, 2004). Experi-ments done in Xasks and in 15- and 150-L reactorsshowed the beneWcial eVect of spent yeast on S. melilotigrowth (Table 6).

6.3. Dewatered sludge as a carrier

Dewatered wastewater sludge has been tested as a car-rier for S. meliloti by Ben Rebah et al. (2002e). Comparedto the control carrier, that is peat inoculated with YMB-grown S. meliloti, dewatered sludge showed optimal poten-tial as a carrier for rhizobia, because it supported rhizobiasurvival at an acceptable level. Sludge can maintain desiredrhizobial populations, a pH around neutral and an accept-able water holding capacity after 130 days of storage at25 °C (Table 7). Stored at 4 °C, sludge behaved similarly,maintaining populations comparable to those obtained at25 °C. Generally, the viable cell number varied between 107

Table 7Survival of S. meliloti and characteristics of sludge-based inoculants (pre-pared by mixing YMB-grown or sludge-grown S. meliloti with dewateredsludge) after 130 days of incubation at 25 °C (adapted from Ben Rebahet al. (2002e))

Inoculants Characteristics

Carrier Growthmedia

Viable cellcount (cells/g)

pH Moisture(%)

Dewatered sludge Sludge 2.5£ 107 7.70 39.9Dewatered sludge YMB 1.0£ 108 7.80 42.0Dewatered sludge + peat Sludge 1.0£ 108 7.52 35.9Dewatered sludge + peat YMB 2.0£ 108 7.36 39.5Peat YMB 7.7£ 108 6.87 36.4

and 108 cells/g which is higher than the standard of 107 cells/g used in many countries. Moreover, sludge-based inocu-lants can meet the Canadian standard (based on numberviable cells per seed) because rhizobial counts are higherthan the quantity (cells/g) needed to provide the requirednumber of 103 rhizobia per alfalfa seed (e.g. 2£106 per g,which is an example given in Section 3). Sludge is also non-toxic, permits growth after incubation, and constitutes anavailable and low cost raw material. These characteristicsmake sludge a suitable carrier for producing solid-basedinoculants.

6.4. Nodulation capacity and symbiotic eVectiveness of rhizobia in sludge-based inoculants

It is important to note that rhizobia produced under allgrowth conditions in diVerent types of sludges maintainedtheir nodulation capacity (Ben Rebah et al., 2001,2002b,c,e; Dufresne, 2004). Nodulation indices (nodule size,color and number) were determined on legumes inoculatedwith sludge-grown rhizobia. The nodulation indices of S.meliloti (nodulating alfalfa), R. leguminosarum bv. viciae(nodulating pea), B. japonicum and B. elkanii (nodulatingsoya) varied slightly among species and with the origin ofsludges, but were generally close to those obtained forYMB-grown rhizobia. Also, the symbiotic eVectiveness ofsludge-based inoculants (liquid and solid) was compared tothat of rhizobia grown in the standard medium (YMB)and/or stored in peat (Ben Rebah et al., 2002c). Alfalfaplants were inoculated with either liquid or solid S. melilotiinoculants and grown in pots under controlled conditionsin two soils types (clay and sandy soil) previously cultivatedwith alfalfa and containing indigenous rhizoba (Table 8). Inthe sandy soil, solid sludge-based inoculant allowed thehighest alfalfa shoot dry weight while in the clay soil, therewas no signiWcant eVect of inoculation ; however, all typesof inoculants increased alfalfa nodulation in both soils, aswell as rhizobial populations. For example, for the sandysoil, inoculation increased the nodulation indices of alfalfafrom 4 (control) to 8 and 12 and the number of rhizobiafrom 103 cells/g to 106 cells/g. The same tendency wasobserved for the clay soil. Therefore, inoculation of alfalfawith sludge-based S. meliloti inoculants is as eYcient asinoculation with standard inoculants.

Table 6EVects 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)


7. Conclusions

This review has considered the use of waste in the produc-tion of liquid and solid rhizobial inoculants. Many agro-industrial wastes showed good potential to replace the stan-dard medium and the commonly used peat in inoculant pro-duction. Although availability, transport, cost and treatmentof these wastes may limit inoculant production, their use stilloVers a valuable alternative for recycling wastes that sup-ports sustainable agriculture. The use of industrial andmunicipal wastewater sludge remains a challenging but newoption for sludge disposal and recycling, and may reduce theproduction cost of inoculants. Compared to other wastes,sludge is an abundant raw material for inoculum prepara-tion. Sludge can be used without any treatment processbecause of its organic composition (C, N, P, solids) and itsnutrient value. In some cases, sludge pre-treatment may benecessary to maximize cell numbers and to reduce fermenta-tion time. Improvements in sludge technology (formulationof the product, test in the Weld) are still necessary beforelarge-scale application of this new strategy can be realized.

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Agro-industrial waste materials and wastewater sludge for rhizobial - 2007

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