Indian Journal of Experimental B iology Vol. 4 1, Scptember 2003, pp. 1030· 1045

Bioremediation: An important alternative for soi l and industrial wastes clean-up

Carlos R Soccol l' , Luciana P S Vandenberghe I , Adenise L Woiciechowski I,

Vanete Thomaz-SoccoI2, Cristiane Tagliari Correia ' & Ashok Pandei

I Biotechnology Proccsscs Laboratory , Department of Chcmica l Enginecring, Fedcral Uni vcrsity of Parana, PO Box 190 I I , CEP 81531-970 Curitiba - PR, Brazil

2Patoloy Dcpartemcnt. M olccu lar Parasi toly Laboratory, Federal Univcrsi ty of Parana, CEP 8153 1-970 Curiti ba - PR, Bra

.lBiotechnology Di vision, Rcg ional Research Laboratory, CS IR , Tri vandrum, 695 019, India

Industri al and environmental biotech nology are going to new path s, resulting in processes w ith "c lean technologies", with the maximum production and the less residues. Technologies of remed iation and biorcmed iation are continuously being improved using gcncticall y mod ified microorgani sms or those natu ra lly occurring, to clcan residues and contaminated areas from tox ic organ ics . Bioremed iation of soil s, water and marine envi ronmcnts has many advantages but at the same timc it is a challcnge for thc rcscarchcrs and cngineers. Consequently, it is cxtrcmcly important to carry out fcasib ility study bascd on pilot-testing beforc starting a rcmed iation project in order to determinc the bcst conditi ons for the process. The arti cle presents a brief rcv icw of bioremcd iat ion including the descripti on of the diffcrent mcthods applied to soil and industri al wastes, and. finally, somc ex pcricnccs of solid-statc fermentation in relati on to biorcmediat ion.

Keywords: Biorcmcdiation, Cassava bagassc, Coffee husks, Indust ri al waste managcment. Heavy meta ls, Hydrocarbon­degrading, O il shores, Soil contamination, Sol id-state fermentati on

The advances in technology have sustained our industri al ized soc iety. Duri ng the twentieth century, the exp losive development of chemi cal industries has produced a great vari ety of chemical compounds that have led to the moderni zation of our lifesty les. The large-scale producti on of a variety of chemical compound s, however, has caused global deterioration of environmental quality t. Contamination of soil s, groundwater, sediments, surface water, and ai r with hazardous and toxic chemicals are serious problems which have been faced by our world tOda/ . Land degradation is a lamentable problem which is increasing continuous ly all over the world. A third of Europe's 300 million hectares of drylands suffer from deserti fication and the ensuing reduction in biological and economic productivit/. Pesticide and fertil izers are major sources of pollution followed by industri al processes, waste and waste water sludge di sposal, and acc idental release4

• Once an area is polluted, the next step 1 is to suggest possible corrective actions. Over the years, many methods have been tested, used, approved or rej ected. Some conventional methods can be cited such as prevention and reduction , reuse, employment of degradable material s, recycl ing, incineration , pyrolys is and landfill .

* For correspondencc: Fax: 0055 (4 1) 36 1 3654, E-mail : socco l @ufpr.br

The concepts of biotechnology are not new. Fermentation, the most common form of biotechnology , is known and ex perienced when molds were used to produce ferme nted food in China, and beer and bread were made in Egypt5

.6

. The fi eld of biotechnology can be divided into four segments: biomedical, agricu ltural, environmental and industri al. The biomedical segment is growing a lot, including the production of antibody-based di agnos tic test kits , vaccines, aimed by the genetic engineering techno­logy and the recombinant DNA technology . The agricultura l border has seen big progresses with the geneticall y engineered organisms, which seems to be a very important too l to produce food to human being from undeveloped countries using unhosted areas. Industri al and env ironmental biotechnology are going to new paths, resu lting in processes with "clean technologies", with the max imum prod ucti on and the less residues .

Bioremedi ation is a general concept that includes all those processes and act ions that take pl ace in order to biotransform an environment, already altered by contaminants, to its ori ginal status? It is the naturall y occurring process by which microorganisms either immobili ze or transform environmental contaminants to innocuous end products. Its an important so il and groundwater remedi ati on strategy due to several

SOCCOL el al.: BIOREMEDIATION: AN ALTERNATIVE FOR SOIL & INDUSTRIAL WASTE CLEAN-U P 103 1

characteristics: (a) permanently eliminate contami­nants through biochemical transformation or minera li­zation ; (b) avoid harsh chemical and physical treatment; (c) operate in situ ; (d) be cost effective8.

While advances have been made in understanding many of the basic phenomena underlying bioremediation, such as elucidation of biochemical degradation pathways and identification of indigenous bacteria capable of biodegrading pollutants, it remains diffi cult to engineer fi e ld-scale bioremediation applications. This biological process invo lves complex interactions of biological, chemical and physical processes, and requires integration of phenomena operating at scales ranging from that of the microbial cell (I0·6m) to that of the geological site (10-1 OOOm). By necessity, laboratory investigations of biodegradation are performed at a small scale. Intermediate-scale experiments, with soi l columns for example, are frequently used as a bridge between microcosms and a pilot- or field-scale system8

Several review articles have been publi shed which enumerate the definition, applications of bioremediation and physical , chemical and biological conditions necessary to facilitate contaminant biodegradation 1.2.7. 11.

This work intends to presents a brief review of so il bioremediation, its development, the main factors limiting its use, the characteristics of principal methods of bioremediation with application in soil contamination with pesticides, herbicides, o il (hydrocarbons), food waste and heavy metals. Finally, the experience of the Biotechnology Processes Laboratory - LPB of the Federal University of Parana (UFPR) in bioremediation will be presented.

Bioremediation development Mearns 10 repolted that during 1990s, bioremedi ation

was popularized as the ultimate solution to oil spills. Responders and the public were encouraged by field tests with nutrient applicatio ns at the 1989 Exxon Valdez. oi l spill and other earl y experiments. Most of these proposals offered to add some combinati on of oil-degrading microorga nisms and/or nutrients to o iled shorelines. Although some of the products "worked" in the laboratory, fie ld tests fe ll far short of success or resulted in controversy about the representativeness of otherwi se success ful site-specific results l2. However, throughout the 1990s spills were viewed as opportunities to test favored products and methods. Politicians and media thrived on the public ity. Since then, bioremediation found use in numerous app lications, inc luding clean-up of ground-wa ter, so il s, lagoons. sludges, and process-waste streal11s2

.

The "post-Exxon Valdez excitement" has subsided. It has been replaced by more sober, sc ientifica ll y­valid approaches 10. To understand the scope and limits of bioremediation , it is necessary to understand something about the composition and fate of contaminants and about geologica l characteristics of the site to be treated. As suggested by Mearns 10 there are many works that should be consulted about this theme including Atlas I 3

.14

, Zobell l5, the starting point, Hoffl6, Swannell et ai.17

, Venosal 2 and others which are not easy to obtain internationa ll y.

Bioremediation engineering and methods According to Iwamoto and Nasu l, bi oremed iati on

technolog ies can be c lassified as ex situ or in situ. Ex situ technologies are treatments that remove contaminants at a separate treatment facility. In situ bioremedi ation technologies involve the treatment of the contaminants in the place itse lf which offers great advantages. The main are: can be done on site , e liminates transportation cost, eliminates waste permanently, si te disruption can be minimi zed, applicable to diluted and widely diffused conta­minants and affordabl e. In situ bioremediati on processes are c lass ified into the following categories: bi oattenu ati on, biostimulation and bioaug mentation.

Bioattenuation is the method of monitoring the natural progress of degradation to ensure that contaminant concentration decreases with time at re levant sampling points. Thi s method is widely use to cleanup underground storage tank sites with petroleum-contaminated so il and ground-water in the United States l8.

If natural degradation does not occur or if the degradatio n is too slow, the environment has to be manipul ated in such a way that biodegradati on is stimulated and the reaction rates are inc reased. In thi s case, bi ostimul ation includes supplying the environ­ment with nutrients (nitrogen and phosphorus), with e lectron acceptors (oxygen) and/or substrates (methane, phenol and to luene) in controll ed concentrations. Iwamoto and Nasu l cited that, in Japan, the effectiveness of in s itu biostimulati on by methane inj ecti on into TeE-contam in ated g roundwate r was demonstrated by small-scale fie ld experiments funded separate ly by the Environmen t Agency and by the Milli stry of International Trade and Industry.

The third method , bioaugmentatio n, is a way to e nh ance the biodegradati ve capacities of contami­nated si tes by inoculation o f microorgani sms with the desired cata lytic capabilities . It has been practiced

1032 INDIAN J EXP BIOL, SEPTEMBER 2003

intentional ly for years in a number of areas, including agriculture, forestry and wastewater treatment. The use of bioaugmentation is supported by studies showing the deficiency of indigenous microorganisms in some cases and the apparent enhanced bioremed iation rare after the addition of competent microorganisms, These studies were cited by Vogel ' 9

in hi s paper which debates the efficacy of bioaugmentation. The author affirmed that the reinoculation of so il with indigenous microorgani sms directly isolated from the soil is often included in the term bioaugmentation .

In the specific methods used for bioremediation contaminated soi l are: landfarming, composting, intrinsic bioremediation and sluny bioreactor, presented in Table I , which shows the main advantages, disadvantages and application s of each technology, inc luding re ferences7

.

Th ass ito u and Arvanitoyann is7 reported that

landfarming was probably introduced into sc ientific literature by an article published by Dibble and Bartha20 which described disposal by biodegradation of oi ly sludges in soil. Landfarming re lies o n the principles appl ied in agricu lture and aims at controlling the bi ocycling of natural compounds. Generall y, this method can be optimized by the di lution of contaminated soil with clean so il, tilling of the soil to reduce initial toxicity and control of physical parameters, such as aeration, pH, soil mo isture content and temperature .

Composting is a biological aerobi c decompositi on of organic materi als in which conditions are str ic tl y contro lled in o rder to he lp thermo phi lic mi cro­organ isms, present in the so il , to trans form organic materials into a stab le, soil-like product. However, in some cases the deco mpositio n rates a re very slow. For industrial purposes, its necessary to op timi ze microbia l growth in order to increase these rates. This

Table 1- Bioremediation methods accordi ng to Thass itou and Arvanitoyanni s7

Techno logy Princ ipl es Adva illages Di sadvantages Appl ica t ions

Land farmi ng Sol id-phase treatment Simple proced ure. S low degradation rates. Surface cunlarllinali o n. sys tem for conta minated Inexpensive Se lf-heating. Residuc contamination Aerob ic process. Low to so ils: may be done ill situ often re moved . Hi gh medium eo nl amination o r in a construc ted soil exposure ri s \-.. s . May levels. trea tmelll cell . req uire long incuba ti on

per iods.

Compost ing An aerobic mi crobia l More rapid reaction Need bulking agents. Surface cOlllamin ation. driven process that rates. Inexpensive. Se lf- Requires aeration. Aerobi c process. converts solid organic heating. Nitrogen addi tion often Agricultural and human wastes into stab le , necessary. High ex posure was tes . Sewage s ludges, sanitary, humu s-l ike ri sks. Res idual indus tri al wastes, ya rd materia l contaminati on. Incubation wastes, m unic ipal so lid

periods a re months to waste. years.

Intrinsic bioremediation Relies on the natura l Rclati ve ly inex pensive. Low degradatio n rates. Deep cOlllamination. ass imilative capac ity of Low ex posure ri sks. Less contro l over Aerobic o r nitrate the ground to provide Excavat io n not required . environmenta l reduc ing condition s. site remediation and parameters. Needs good Low to medium contro l contaminant hydrogeological s ite con tam inati on levels.

mi grati on. charac te rization. Oil s gaso line. Incubatio n periods a re C hl orina ted aromat ics. months to years. Chlorinated

hydrocarbons.

Sl urry bioreactor Soi l and water ag ita ted Good contro l over Hi gh capital out lay. Surface contami nat ion.

together in reactor. parameters. Good Limited by reactor s ize. Recalc itrant compounds. microbe/compound High exposure ri sks . Soil that binds contact. Enhance compou ndti ghl y. desorpt ion of compound Aerobic and anaerobic from soil. Fas t processes . degradat ion rates. Inc ubation periods are days to weeks.

'f

SOCCOL et al.: BIOREMEDI ATION: AN ALTERNATIV E FOR SOIL & INDUSTRIAL WASTE CLEAN -UP 1033

means optimizing oxygen concentration , pH, moisture content, carbon to nitrogen (C:N) ratio and parti c le size. The compostjng process is initiated by mesophilic bacteri a, which are biologically acti ve at temperatures between 30° and 4SOC. Heat -i s produced after the degradation of organic matter, consequentl y the temperature increases to 50°-60°C. At this range of temperature thermophilic bacte ri a grow eas il /. Composting can be used as a method to stabili ze and decrease sewage sludges, industrial wastes , yard wastes, municipal wastes and, more recently , explosives2.

In situ bioremediation is a natural process occurring ever since the first microbes and excess organic matter were both present in the soil 21. This method expl oits natural ways of recycling nutrients through the cycles of nitrogen and carbon. The main advantage of in situ treatment is that no excavation is needed and no special equipment is required. Thus, it is ideal for treating rocky o r underground water areas. The decomposition of the contaminants is carried out by the indigenous mjcroorganisms which grow on this contaminated soil and can o nly survive in that environment by using contaminating substances as source of energy. Since in situ bioremedi ation is a slow process it may not be a good alternati ve if immedi ate site c lean up is required . In some cases, the metabolic process of degradati on produces undesir­able by-products, which could be tox ic7

.

In slurry bi oreac tor treatment systems, the contaminated soil s are excavated and mixed with water to form a slurry that is mechanica ll y aerated in a reactor vessel. The reactor contents are ag itated to

promote breakdown of soil aggregates, enhance desorption of contaminants from soil solids, increase contact between the wastes and microorgani sms, and enhance oxygenation of the slurry22. Different substances such as surfactants, di spersants and materi als supporting microorgani sms growth, are added to slurry to improve the treatment of contaminated soil and increase the biodegradation capability (United States Environmental Protectio n Agency, 1990 cited by Thass itou and Arvanitoyanni s\ A typi cal slurry can only functio n under the foll ow ing conditio ns: additi on o f oxygen, nutrients and supplemental bacteri a and regul ation of the temperature and pH in order to maintain the microorganism growth and acti vity. Thi s process have a higher cost than in situ bioremediation because of the high degree o f eng ineering in volved. On the other hand, the biodegradation rates o f the same compound are faster in slurry bioreactors compared to the ones obtained by the in situ method7

.

Sturman et a/.8 showed the engineering of bioremediation systems is aided by defining three scales of observatio n: micro- , meso- and macro-scale, as illustrated in Fig . I . The scales definiti on are arbitrary, but serve as a use ful conceptual struc ture fo r approaching the engineering scale-up problem. The micro-scale is taken as the scale at whi ch chemi cal and microbio logical species and reac ti ons can be characteri zed independently of any transport phenomenon . The author presents some examples o f microscale features which are: the composition o f mi crobial consorti a and the kinetics and stoichi ometry

INJEC T ION RECOVERY

'" o ~ c

'" u c o

U

c

Fl u id N u t r ien t s

V> C o

S u iJ s t " ,tes ~ - - - ~ C S u s pen de d ~ C e ll s _~ 0

u

Fig. I - Schematic diagram of the scales of observa tion (Sturman el al x).

1034 INDIAN J EXP BIOL, SEPTEMBER 2003

of transformation reacti ons. The phys ica l scale of these phenomena is the dimension of the microbial ce ll , on the order of 10'6_10'5 m. The meso-scale is defined as the scale at which transport and system geometry are first apparent, w ith the exc lusion of advecti ve or mixing processes. M esoscale phenomena include diffusion, sorption and interphase mass transfer. Possible phys ical scales for mesosca le phenomena include the size of pore channels or soi l particles, the characteri sti cs di ffusion length , or di mension of microbial aggregates (l 0'5 _102 m). Advec tion, dispersion and geo logic spatial hetero­geneity are examples of macrosca le phenomena. The corresponding physical scale for these phenomena ranges from - 101 to _ 102 m or even larger. Many of the phenomena innuencing bioremed iation were presented by scale in the work of Sturman eT al.8.

Phenomena are classi fied accordi ng to the smallest scale at which they can be observed.

In situ bioremediation is a complex undertaking which requ ires an understanding of many physica l, chemica l and biological phenomena. Studies made at micro- or mesoscale may not necessaril y appl y at the field (macro) scale. Observed contaminant loss rates, for example, depend on sca le. Field measured half­li ves tend to be 4- 10 times longer than laboratory­determined va lues, due to scale-dependent rate limitati ons. For a gi ven set of environmenta l conditions there is a single phenomenon which will limit the rate at which bioremed iati on can proceed. Bioremediation engineering must consider all relevant phenomena to determine which w ill limit contaminant biotransformation rate for a particular site. Field sites are typicall y heterogeneous, which can cause different phenomena to limit biotransformation rates across the site. Se lec ti on of remedial strategy should include an assessment of its effects on biotransformation rate­limiting phenomena. Th is will be useful for determining: a) the potential for successful bioremediation; b) whether the rate can be enhanced; c) how to best engineer the process; and d) how to vcri fy bioremed iat ion has occurredS.

Factors limiting bioremecliation technologies A bioremediation process is based on the acti vities

of the aerob ic heterotrophi c microorganisms. The success of bioremediati on depends on hav ing the ri ght microbcs in the right place w ith the ri ght environmental factors for degradation to occur. The right microbes are bacteria or fungi, wh ich have the phys iologica l and metabol ic capab ilities to degrade

the pollutants. Microbial activ ity is affected by a number of physica l-chemica l environmental parameters. The factors that direc tly impact on bioremed iati on are energy sources (elec tron donors), electron acceptors, nutrients, pH, temperature and inhibitory substrates or metabolites. It is also important to observe the distinctions between surface soil s, vadose zone so il s and groundwater sediments which is the content of organic materi al. Surface soil s, which typ ica ll y recei ve inputs of organic material from plants, wi II have higher organic matter content. Subsurface so il s and groundwater sediments have lower levels of organic matter and thus lower microbi al numbers and popu lation diversity than surface soi ls. Bacteria become more dominant in the microbial community w ith increasing depth in the soil profile as the numbers of other organi sms such as fungi and ac ti nomycetes decrease2

.

Table 2 presents the main factors affecting bioremediation. The effecti ve energy source for an aerobic heterotrophi c organism is a funct ion of the average oxidation state of the carbon in the material. In general , higher ox idati on states correspond to lower energy yields which thus prov ide less energetic incentive for microorganism degradation. The outcome of each degradati on process depends on microbial (biomass concentration, population diversity , enzyme activities), substrate (phys ic­chemica l characteri st ics, molecular structure and concentrati on) and environmental factors (pH , temperature, moisture content, Eh, availab ility of electron acceptors and carbon and energy sources)2

Concerning the bioavailab ility , the rate at wh ich microb ial ce ll s can convert contaminants during bioremediat ion depends on the rate of contami nant uptake and metaboli sm and rate of tran sfer to the cell (mass transfer). Increased microb ial conversion capaciti es do not lead to higher biotransformation rates when mass transfer is a limiti ng factor2

. The decrease of the bioavailab ility in the course of" time is often referred to as aging or weathering . It may result from: a) chemical ox idati on reactions incorporating contaminants into natural organ ic matter; b) slow diffusion into very small pores and absorpti on into organic matter; and c) the formation of semi -ri gid films around non-aqueous-phase liquids (NA PL) with a high resistance to ward NAPL-water mass transfer. These problems can be overcome by the usc of food­grade surfac tants13

.

-t

SOCCOL el af.: BIOREMEDIATION: AN ALTERNATIVE FOR SOIL & IND USTRIAL WASTE CLEAN-UP 1035

Table 2- Factors affecting bioremediation according to Boopath /

Microbial

Growth until critical biomass is reached Mutati on and horizontal gene transfer Enzyme induction En richment of the capable microbia l popu lations Producti on of toxic metabolites

Ell llironll1elllal

Depletion of preferential substrates Lack of nutrients Inhi bi tory env ironmental conditions

Subslrare

Too low concentration of contaminants Chemical structure of contaminant. Tox ici ty of contaminants Solubi lity of contaminants

Biological aerobic liS anaerobic process

Ox idat ion/reduc tion potenti al Avail ability of electron acceptors Microbial popu lation present in the site

Crowlh subs/J'are vs cO-/llelabolis/ll

Type of contam inants Concentrat ion Alternate carbon source present Mi crobial interaction (compet ion. success ion and predation )

Physico-chemical bioa vailabilil), of polluwllls

Equ ilibrium sorpti on. Irreversible sorption Incorporation into humic matters

Mass lrall.ller lilllilaliolls

Oxygen diffu sion of solubi lit y Diffusion of nutrients Solubility/mi sc ibility in/w ith water

Applications of bioremediation methods

Contaminated soil d· ?4 Accor mg to T roque t, et al. - , the re a re at least 350

000 contaminated sites a llover west Europe, and the bioremediation of only the ri sk iest of these sites w ill consume more or less 440 billion Euros. The major responsible fo r this po llution are petroleum hydro­carbons based products. Recalc itrant compounds contaminating so il s is a very bi g problem widespread around the wo rld , especiall y in industrial areas, where these organi c compounds are degradated very slow ly or are not in the natura l so il environment. It happens for man y reasons, such as, the low temperature ,

particularly in countries w ith cold cl imate, the difficult in keep aerobic conditions, some times necessary to specific degradations, the lack or poor nutrient balance, with low concentrations of nutrients, the non homogeneous di stribution of the contaminant allover the site, the lack of the ri ght way to degradate the recalcitrant compounds present at the contami­nated so il , because, the suitabl e pathway natura l or op timi zed must be set according to conditi ons defined in laborato ry' I . Bioremedi ation appears to be a promising technol ogy to deal with so il and subso il contaminated with organic substances. These organ ic carbo n compo unds are used by microorgani sms as energy source and for biding cell s blocksn. Xenobiotic compo unds contaminating so il and grou nd water can suffe r mi crobi al biodegradat io n, th rough the process ca lled bioremediat ion, according to the laws of thermodynami cs. Studies are being done, using higher pl ants to provide carbon and energy sources to keep the mi crobial population in a balanced medi a ideal to optimi ze the contaminants biodegradation25

.

Bioallgmel1tatioll Bioaugmentation is a process where an ideal o r a

poo l of microorgani sms o r enzymes or a specia l degradating agent is added to a volume of contaminated so il o r water, in order to prov ide the effic ient degradati on o f the pollut io n presented at the site, or enhance an spec ific biological acti vity. it is difficult to predi ct the e ffect of bioaugmentation in soi I bi oremediat ion, because of the di versity of the microorgani sms required, the envi ronmental heterogeneity and because of the variation at the degradation response to in front of c ritica l parameter such as humidity , nutri ents ava ilable, temperature, etc. 18

. Thi s process has been used in so il s for years to recover degraded areas in ag ri culture26

, fo rests and wastewater treatment27

. Studies support th at bioaugmentation is an usefu I process at thc treatment of po lluted s ites, where the natural OCC UIT1I1g

microorganisms show an inability to enhance biorcmedi ati on rates. So, the addition of competent microorgani sms apparentl y improve the process of bioremediation. Often the bioaugmentation is done with the polluted site re inoculation with indigenous mi croorgani sms isolated from the same so il , but in big quantities. Bioaugmentation, to treat contaminated soils, is be ing subject of many studies, and its effecti veness is not proved. The subject must be yet di scussed l8

.

1036 INDI AN J EXP BIOL, SEPTEMB ER 2003

Bioremediation of industrial residues All industri al process ing generates res idue, and a

few decades ago the cha llenge res ided in speed up the industria li zation process. Nowadays the attentio n is to find ways to deal with the grow ing industri ali zati on and the problems assoc iated with it. The big amount of industri al res idues generated by the industries causes great probl ems with the di scharging o f solid and liquid wastes, as far as water resources. A crucial problem is the land degradati on a ll over the world. Solid wastes , because of its vo lume and not o nl y fo r its hazard , are probably the most problematic and its f inal deposition can produce large po lluted areas. The steps to solution the problem pass tho ught pre vention and reductio n of the solid waste vo lume, reuse, recycling, inc inerati on and landfill , and a lso the use of modern and innovati ve methods like des igned composting and bioremedi ati on 7

Fruit and vegetable industries Industries that processes fruits and vegetables are

very important part of the food industries, because thi s market is inc reas ing and innovating day by day, includ ing processes li ke canned, froze n, dehydrati on, drying frui ts and vegetabl e, f ruit pulping, juice and concentrated. However, as consequence of these inovati on and grow ing, the generati on of residues is also increas ing. In genera l, was tewater fro m fru it and vegetable processors, contain s main ly wate r soluble organi c compounds, but sometimes it a lso has a large amount of suspended solids, with a hi gh bi ochemi cal oxygen demand (BOD) and reactive when in contact with microorganisms in general. The chemi cal characteristi cs of the wastewate r, such as pH, BOD, tota l suspended so li d (T SS), di ssolved oxygen (DO), varies large ly, depend ing on the ki nd of vegetab le or fruit processes, the techno logy and quantity of water

used . Some exampl es of wastewater characteristi cs are shown in T able 3 . pH can be a serio us prob lem at th is industries, because some processes of peeling methods need high p H. The nitrogen content of fruits and vegetable wastes occurs in various forms, the majo rity as complex organic ni trogen, sometime di ffi cult to break down. So the lack of ava il ab le nutrients, especia ll y nitrogen is his torica ll y a prob lem in fruit and vegetable wastes28

.

Bes ides these compo nents, the was tewate r can contain vari able q uantities of herbic ides, pesti c ides and cleaning chemi cal, what urge fo r carefu l treatment. The solid waste fro m fruit and vegetab le industries can be treated with composting or land fa rming, with prev io us removal of water, and adj usting of the pH, to ensure best conditi ons to the microbi al growth . Sometimes a bul king agent, such as straw, paper, mature compost, coffee res idues or by prod ucts of its own production, can be added to improve the bed porosity, whi ch he lps even in the

d · 7 water ramage.

Meat and poultry industries Thi s kind o f industri es is o perating a ll over the

world and has a central ro le in food chain producti on . Great di ffe rences occ ur from one nation to another, and they re fl ect in the organization, perfo rmance, quality, uses and d iets of loca l meat or poul try process ing. Because of these di ffe rences, the so li d res idues and wastewater of the meat and poul try products industries vari es a lot. In general, fo r the liquid water, the organi c matter can be deg raded by microorgani sms in aerobi c conditi ons, without any nutrient supplementat io n and a prev ious inoculation is not necessary , because there is a natural microorganisms community that prov ides the organic matte r degradatio n. The Total suspended so li ds is

Table 3 - Charac teristics of the was tewater generated in some fru it and vegetable industries

Crop

Apples Carrots Corn Grapes Mushrooms Peaches Pickles Potato ch ips Potato sweet Tomato pec led

Flow (I/ton of raw product)

10 .0 14 .0 80 6.0

33.0 13 15.0 7.0 9.0 0.9

Source: Barnes e f (II. 2X

BOD (kg/ton of raw product)

9.0 15.0 13.5 4.5 7.0

17.5 2 1.0 12.5 46.5

4.7

TSS (kg/ton of raw product )

2.2 8.5 5.0 0.8 3.6 4 .3 4 . 1

16.0 28.0

6.0

SOCCOL ef al .: BIOREMEDIATION: AN ALTERNATIVE FOR SOIL & INDUSTRIAL WASTE CLEAN-UP 1037

sometimes hi gh, but can be easily removed. The same happens to the fat , oi Is and greases content, that is a characteristic of this kind of liquid residue. The removal of this solids before the liquid treatment and a posterior specific treatment of the solid residues is important. A big amount of ammonia can be found in most meat and poultry liquid and solid wastes, because of the breakdown of prote inaceous waste into amino acids and ammonia, that is directly toxic to many organisms. Microorgani sms that can cause diseases to human and animals are often found in meat, poultry and rending wastes , such as the bacteri a of the genus Salmonella . Because of the big differences found at the industries process ing and technologies used , a very careful characterizatio n of the solid and liquid waste is a lways recommended in order to evaluate the problem and suggest the right solution for each case28

.

Beverage and drinking industries The consumption of beverages is increasing greatly

since 1 970s, and so the pollution caused by the beverages industries during the processing and bottling of the beverages, in particular this fermentation industry , that can be divided into three categories: brewing, di stilling and wine manufacture, each one with its own characteristics, producing different kinds of contaminants, but having in common a high BOD. So the difficulty in treat residues of these fermentation industries (such as, brewing and winery) , is the flow and organic load of the wastes. Since the industrial effluent has hi gh quantities of tannin s, pheno ls and organic acids, the anae robic treatment results in higher performance. For distilleries, the amount and organic load of the wastes vary according to the raw material used . Using molasses, the load is three time that of rai sins. The res idue of fermentation process, the vinasse, which is always present, needs a very accurate and long biological treatment for some days , in order to reduce its chemical and organic load7

.

Dairy industries This kind of industries contributes substanti a ll y to

the water and soil pollution all over the world. Dairy industries goes from the milk production , to the milk industries, such as , butte r, anhydrous milk , cheese, casein, condensed and evaporated milk , dried milk , whey processing, ice-cream and frozen desserts, yogurt , and other milk derived . The dairy waste is normall y very high in organic matter which is

composed basically of fatty substances, prote inaceous material and sugars, and largely varies in quality and quantity, depending on the kind of industry and the final product. Present considerable variation in pH, from 4.0 to 9.5 ; a rel atively large load of suspended solids, from 400 to 2000 mg/L, and present a large variation of waste supply . Dairy wastewater ' may contain also various kinds of cleaning chemicals, mainly detergents, used to di ssolve and remove proteins and eliminate fats through saponification. The use of these chemical compounds hardly influences the total organic matter present at the wastes, measured by Chemical Oxygen Demand (COD), and that difficult in their treatment. The effect of all these chemicals must be analyzed . Because of the great range in quality and quantity, many types of aerob ic and anaerobic processes can treat wastewater of dairy indu stries. Activated sludge process can be used and achieve partial denitrification and some uptake o f phosphorus. In laboratory scale, removal s of 90% at the COD was achieved us ing anaerobic sequencing batch reactor, with a sy nthetic milk substrate7.28 . Industrially, the wide variation in characteristics of dairy plants wastes require special consideration in design of treatment plants, mainl y when it comes to bioremediation. The wide variation in volume, fl ow rates, organic strength and composition during the day , from day to day and throughout the dairying season, temperature, pH, concentration of milk fat, and settle able solids, nutrients, and other, alter greatly the needed treatment and the act ions to recover a polluted site. Dairy process ing wastes are particularly suitable for di sposal onto land. The balance mineral profiles, the presence of nitrogen, the lack of toxic substances, and the irrigati on benefits turn thi s wastewater able to use of land di sposal in some countries. Thi s technique provide a high treatment efficiency, using the natural so il occurring microorganisms, need low capital and operation costs and provide a return in fert ili zation of the soil. The onl y requirements are large areas, sometimes far from the industry and properties environmental temperatures . Some times, special inoculants wi th properties microorgani sms can be added to the wastewater in order to accelerate the biological trea tment. When improperly designed and operated, the process can cause odors, pounding, pasture, and attract flies and insects. Aerobic biochemical treatment includes activated sludge, oxidation ditches, lagoons, trickling filters and rotating biological contactors. Anaerobic biochemical treatment are some

1038 INDIAN J EXP BIOL, SEPTEMBER 2003

times suitable, what includes anaerobic lagoons, but it b f · ~ appears to e or use III pretreatment systems- .

Bioremediation of oil residues As a result of industrial act iviti es a wide va riety of

tox ic organi c chemicals are being put acc identally or de liberately into the environment , and petrol eum hydrocarbons are one of the most important. Oil residues bioremediati on is bei ng studi ed for at least hal f a century, but the success of the process is not yet very confirmed in Illany situations. It seems to be a su itable and necessary alternative because especiall y marine environment is being target of contamination from va rious sou rces. In quant itative terms, contam in ation with crude oil is among the most important organi c pollutants in marine and soi l environments, as a result of human ac tivity that release from industri al install ati on, transportati on, storage, discharge from effluent treatment and lots from terrestria l sources. As it is known that the maj or components of crude oi I are biodegradab le, bioremed iati on is a technique suitab le fo r the recovering of the environmental. The biodegradation is limited by ava il ab le of nutrients such as nitrogen

I(P9 30 E b ' I" d b I and phosphorus .-. . . ven elllg Imlte y severa facto rs, such as, the presence of oxygen and su itable nutrients, the abundance of hydrocarbon-degrading, . and the presence of the right microorganism in a proper quant ity, the degradati on of the components of the oil happens in the environment , as a result of the act ion of mi croorga ni sms such as bacteria, fungi and yeasts , that can degrade natura ll y, oil components. So, the application of oil -spill bioremediati on of so il and

water contaminated can be considered an ava il ab le technology, but because it 's empiri cal development one faces with difficu lties in formu lating treatment strategies to produce a specific result in terms of degradation rates and residual contam inati on concentration29 Gasoline leakage from underground tanks at gas stati on and from pi pel i nes, as far as marine spill s are reaching alarming rates produc ing every day sources of water and coastal co ntamination, threaten all forms of natural li ves . Petroleum is composed of a mixed of compound that can vary in quality and quantity, depending upon the oil source, age and geologica l his tor/o. The pri ncipa l classes of components of the crude o il can be sum mari zed as show n at Table 4. The pol yaromati c hydrocarbons (PHA) associated with the oil contami nation is known to be carcinogens, mainly benzopyrene, the most tox icJo. Microorgan isms responsible for the degrJdJtion of hydrocarbon compounds are widely spread in marine, fresh water and so il hab itats, including bacteria, fung i and yeasts. The degradation of any kind of hydrocarbon contam in ant. since crude oil , to refined oil , gaso line components, etc, in vo lves a consorti a of microorganisms, in both , eukaryot ic and prokaryotic forms, and man y genera is responsible fo r the degradati on, as shown in Table 5.

Among the oil components, n-alkanes with 10 to 20 carbons at the chains, even being more toxics, are preferred substrates, and are more qui ckly degraded. Waxes, or long chain alkanes, with 20 to 40 carbons at the chain , are hydrophobic solids, and due to its poor water solubility are difficult to degrade. Branched chains are more di fficu lt to degrade than its

Tabl c 4 - Componcnts of crucle oi I

Petroleum Hydrocarbons

Saturate Compounds

Aromatic Compoll nds

Resi ns

Asphaltcncs

Source: Balba ('{ 01. .l()

Classificati on

Stra ight Chai n A lkanes (normal alkancs) Branchcd A lkancs ( isoalkancs)

Cyc loalkanes (naphtcnes) Monoaroilla tic Hydrocarbons (Volat il e fraction) Polyaromati c Hydrocarbons ( aphthenoaromati cs) Aromat ic Sulphur Compounds Amorphous solids (Tru ly disso lved in thc oi l) Large molecules (Colloida ll y dispcrscd in the oil )

Examples

Ethanc. Ethcnc, Propanc. Paraphinics. Waxcs.

Isobutane

Cyc lohexane Benzene, Xy lenes, Tolucnc. Naphthalcnc, Antraccnc. Phenantrcnc Th iophenes, Dibenzoth iophcncs

Polar macromolccu lcs containing Nitrogen , Sulrur and Oxigen.

SOCCOL et al.: BIOREMEDI ATION: AN ALTERNATIVE FOR SOIL & INDUSTRIAL WASTE CLEAN-UP 1039

Table 5 - Main microorgani sms able to degrade hydrocarbon compounds

Ki nd

Bacteri a

Yeast

Fungi

Source: Balba, et al. 3o

Genera

Noca rdia. Pseudomollas, Acinetobacter, Flavobacterium. Micrococcus, Arthobacter. Corynebacteriul'. Rhodococcus, Alcaligenes. Mycobacteriun, Bacillus. Rhudotorula, Candida. Sporobolomyces Aspergillus. Mucor, Fllsarillm. PeniciliulII, Phanerochaeta

corresponding normal alkane. There is a especial microorganism specie to better degrade a celtain o il component, such as aromatics, po lyaromati cs (PH A), whjch degradation depends on a seri es of factors, such as, the molecular weight of the compound, and the establishment of a degradation rate with the synthes is of intermediates, paJticularl y dihydrodiols, that can be much more toxic than the orig inal compound . But fortunately studies with the sediments, suggest that these compounds are rapidly transformed, what avo ids its accumulation3D

.31

. Cycloalkanes have slower degradation rates than normal alkanes, and this degradation in vo lves many microorganisms species. Aromati cs condensed and cycloparaphinic structures, tars and asphaltic materials are very res istant to biodegradation. Asphathenes are products of petro leum that appears to have inert characteri sti cs, such as insolubility, and aJ'e resistant to microorganisms attack, so it is suggested to be environmentally not hazardous29

. According to Balba el al.3D

, many technologies are available to do the decontamination of polluted environmental, such as, excavation and containment in safe landfills, bio logical stabilization in situ , soil washing, solvent ex traction, desorption, vitrifi cation and incineration. But even with the cost and the lack of effecti veness at the destruction of the contamination, environmental bioremediation appears to be one of the most promjsing technologies for dealing with environmental contamjnants, because of the wide range of contaminants that can cause environ­mental pollution, bes ides the bioremed iation techno­logies simulates natura l processes, and it is less expensive than the equi va lent phys ica l-chemical process to do the same job.

Bioremediatioll of xenobiotic compounds As a resul t of a large-scale produc ti o n of a vari ety

of chemi cal compounds, happened a g lobal dete rio ra-

tion o f the environmenta l quality. Parti cul arl y the xenobio ti c compounds that are diffe rent in structure of the other natura l organi c compounds , such as po lychlorinated bipheno ls (PCBs), thrichloroethylene (TCE), perchloroethy lene (PCE), and so on, are the chemi cal compounds with hig h tox ic ity, res istant to biodegradation and once o n the so il , can be very haza rdous via web food , so contaminati on of soils and ground wate r w ith these organic compo unds is a c riti ca l and a present problem nowadays l.32 . Genera lly the concentration o f pesti c ides in the environmenta l is low and contro lled naturally, by the presence of many a lte rnati ves in a complex natural medi a where they are degradable at the natural o rig in . However, the so il saturati on and the intox icati on of the so il microorgan­isms can occur and as a result of the systemati c inc rease o f the food production necess ities, what lead to an use of more and more chemical products to face the c rops pests. The so il has some characteri sti cs of sorpti on that depends o n the kind of the so il , and the env ironmental conditions. So it is di fficul t to reproduce these conditio ns at the laboratory, to study the pheno mena, bes ides, the organic po llutant is never alo ne, but assoc iated with o ther compounds, what modi fy the sorption or biodegradatio n response for a specific compound 32

. Bio logica l bi odegradation of organi c compounds, are be ing used for at least a century, to treat sewage, solid waste, industri a l res idues and a series o f man-made organic chemical, the xeno bi~tcs. It sugges ts that the organi c compou nd th at is produced bi o logica lly can be destroyed bi o logica ll y with more or less fac ility. The prob lem with recalc itrant compounds ra ised with the development and use of sy ntheti c dete rgents and pesti c ides that cause harmful effec ts to the environment , part of the problem linked to the ir res istance to bi odegradation. So eng ineers faced big proble ms with these new recalc itrant contaminants th at are di ffi cult to degrade and are spread at nature th at o ffer the lack of co ntro l, the opposite co mpared to engineered reacto rs. What turns a molecule reca lc itrant is bas ica ll y its structure th at prevents the actio n of enzy mes, what make the compound in access ibl e. Besides, tox ic enviro nment and the lack o f some essenti a l growth fac tor le t the natura l occurring microorganisms unable to metabo li ze these compounds because of phys io logical inadequacy. i t suggests that the re is a conjunct of fac tors inc ludi ng the co mpound characte ri sti cs, the environmenta l it se lf and the appropri ate mi croorgani sm with the capability to degrade the chemical present. If one of

1040 IND IAN J EXP BIOL, SEPTEMB ER 2003

these factors were incorrect or inappropriate, must be cOITected to biodegradation takes place. Most natural and xenobiotics compounds are biodegradable by the suitable microorganism through its normal functions for growth and energy production, where the organic material is the plimary carbon source. These conversions can take place in aerobic or in anaerobic conditions, depending on . the palticular microorganism or conversion desired9

. The study of Mattins and Mermoud32 with four dinitrophenol herbicides (2,4 din itrophenol, 2-methyl-4,6-di n itrophenol, 2-sec-buty 1-4,6-di nitrophenol and 2-ter-butyl-4,6-dinitri phenol), showed that these compounds with more or less effi ciency, depending on the compound and the soil , can be removed from the liquid phase (humidity), by sorption at specific sandy soil components. Besides the acti vity of the pool of microorganisms presented at the soil can be responsible for the site treatment. According to McCait /, the selection of the environmental conditions is impOitant to set the treatment process, for example without oxygen, nutri ent needs are normally less, and the growth rates are slower. But this condition is suitable only in some cases. In the case of aromatics hydrocarbon, degradation rates are normally enhanced in aerobic conditions, so the introduction of oxygen can be lIseful. To select a microorganism or a pool of microorganisms can be useful to degrade or bioremediate specific areas, with a specific pollutant. It includes the exposure of aseptica lly obtained soil to the contaminants at ideal conditi ons for biodegradation. If the biodegradation of the contaminant occurs, the microorganisms naturally presented at the so il are able to recover the polluted area. It is necessary provide time and physico-chemical conditions to the natural bioremedi ation . Natural occurring microorganisms at soil or water sources have a physiological versatility and catabolic potential to degrade a large number of organic molecules. Almost all organic or sy nthetic compound, independent of its molecular structure or weigh, can be degraded by one or other microorganism in a special environment, into simple structures or into CO2 and H20. The search for a microorgani sm that can degrade specifics xenobiotics compounds, can be done among those natural occurring microorganisms at the polluted site, or can be done taking advantage of natural or induced gene, transfen'ed to build up hybrid or new degradative pathways. Genetic Engineering can be a useful tool, transfen'ing genes, to speed up potenti al of natural communities of microorganisms, that under appropriate conditions will be able to degrade harmful compounds]3 .

Bioremediation of heavy metal residues It is known that low concentration of heavy metals,

the trace elements, is necessary, and improve the lignolitic enzyme system, in wood-roting fungi. In P. Phanerochaele chrysosporium synthetic cultivation media, low additions of Zn and Cu , increasing the activity of lignin peroxidase and Mn-peroxidase, bes ides metal in solution increase the so lubili za ti on and mineralization of li gnin. It is also related the positive effect of Cli addition at the production of laccase in cu ltures of Ceriporiopsis subvennispora, Trichodenna versicolor, Pleurotus OSlreatus, Ganoderma applanQ1UfII, Pleurotlls sajor-caju, and others. As far as, the role of others traces elements, such as, Cd, Hg, Fe, Co, Ni , Ca, at the metabo li sm of fungi are widely related, but even trace elements in big quantities, can be toxi c. It is known that non­essenti al metals can exert tox ic effects in much lower concentrations. Quantities of more or less 0 .1 to 0.25 mM of heavy metal in the media can affect strongly the microbial metaboli sm. Cd and Hg are the most tox ic metals, lead ing to severe growth inhibition of some white-rot fu ngi like, Phanerochaete chrysosporiul1l . Some metal s are more and others are less tox ic, depending on the kind of microorgani sm tests. Toxicity can occur through a number of mechani sms, such as, ( I) inhibition of some enzyme necessary for the microbial metaboli sm, (2) precIpitation or chelation with some other essential metal, what limit its bioava ilability, (3) catalys is of essenti al metabolites or (4) competition with essential metals acting as antimetabolites. In spite of thi s tox ic effect, the ability of wh ite-rot fungi, to absorb and accumulate heavy metal s, and the favorab le mechani c properties of the mycelia pellets, prov ide an opportunity to the use of these fungus in selective work of removing heavy metal from so il and water34.35 . For years , the intensive use of the so il fo r agricu ltural purposes and the mass ive use of ferti li zers and amendments such as activated sludge and manure containing high amounts of heavy metal, caused the accumul ation of these metal in the soi l. Besides, heavy metal at the atmosphere, due to industi'ial activity sett le onto the soil , increasing the soil pollution problem. The case of cadmium is important because of its hi gh tox icity and mobility in the soil. The metal can be transferred to the pl ant system what may lead to accumulation in roots, stems, leaves and especially in edible parts of the crops36.37 When it is necessary to reduce the heavy metal amount in agricultural so il s, for economical and technical

t

SOCCOL el al.: BIOREMEDI ATION: AN ALTERNATIVE FOR SOIL & INDUSTRI AL WASTE CLEAN-UP 104 1

reasons conventional treatments used in industries cannot be appli ed , phytoremedi ati on is ' the solution. The so il or water bioremedi atio n, are indicated to avoid the transfer of heavy metal to plants or to groundwater, but the so il treatment will take many years, and the area cannot be used for food crops during the treatment. The phys ico-chemical and the phytoremediation treatment consist on immobili zing the pollutant in the soil. Especiall y cadmium adsorption by physico-chemical methods can occur in certain and controlled conditio ns of p H, humidity and temperature38

.39. The other a lternati ve to immobili ze Cd is done by inoculation of the soils with viable microorgani sm, that accumulate big amounts of the metal in soils even being these microorganisms the minor part in mass at the site. After the inoculation of the soil with free microbi al cells, it was showed the reduction of the heavy metal transferred to plants. In some cases, phytobioremediati on showed micro­organisms that accumulate high cadmium levels when compared to phys ico-chemical conditions of so ils, even the experiment carried out with syntheti c ri ch media, very different from real environmental conditi ons. The success of the treatment depends on the survival of the desirable microorgani sms at the soil, where the face the natural soil microtlora and unfavo rable environmental conditions, such as vari ation of pH, temperature,

humidity, so it is necessary a previous cell adaptation to the new conditions4o. Working with low concentrations of cadmium, Lebeau et al.37 reached an accumulation of 69% of the metal presented in solution, with the bacterium ZAN-044, 99.5% similar to Bacillus simplex. At the same experiment, the actinomycete R27 and the bas idiomycete Fumitopsis pinicola, did not reached the same cadmiu m accumulation. White-rot fungi can up take and concentrate heavy metal in their mycelia. Table 6 shows the amount of heavy metal that can be uptake by some genera of white-rot fungi. Different from organic contaminants, metals cannot be degraded, only can be sometimes, altered their chemical state. The mechanism used to accumulate heavy metal by the white-rot fungi is not yet well studied . Other fungi uses to uptake both , the essenti al and the non-essential metal the same transportati on system present in the ce ll membrane. For example in the fungus Paxillus in volutus, Cd uptake, invo lves a rapi d bOllnd with the ce ll wa ll and a transport to the ce ll inside. At the cell 50% of the metal is bound to the ce ll wall , 30% stay at the cy topl asm and 20% are put into vaclloles34

.35 .

Solid-state fermentation and bioremediation Since 1986, the Laboratory of Biotechno logical

Processes (LPB ) of the Federal Uni versity of Parana

Table 6 - Microorgani sms used to test the uptake of heavy metals.

Mi crorgani sm

Pleu/'Olus OSlrealus

Daedalea quercina

Galloderma applanalus

Slerellm hirsUllIm

Volvariella volvacea

Source: Baldrian35

Heavy metal bioremediat ion

Cd Cu Zn Cd Al Zn Pb Cu Al

Cd Pb Ca Al Cd Pb Ca Cu Hg Pb

Accumulation Amount (lg dry weight)

20 mg/g 10 ).lg/g 5 ).lg/g

20 ).lg/g Zn > Cu > Pb > AI

600 ).lM/g

272 ).lM/g

602 ).lM/g

96,6 ).lM/g

Cu > Hg > Pb

Initial metal concentration

150 ppm 5 mM 5mM 3 mM

ImM

I mM ImM I mM ImM I mM ImM ImM

Reference

Favero el aiM Sanglimsuwan el a/65

Sanglimsuwan el al65

Gabriel el al. 66

Gabriel el al. 67

Gabriel el al.67

Purkayastha & Mitra68

1042 INDIAN J EXP BIOL, SEPTEMBER 2003

(U FPR), whose responsible is Professor Carlos Ri cardo Soccol , has been developing a series of research projects for the valori zati on of tropical ag ri cultural products and sub-products in Brazil by solid-state fermentation (SSF)4'.

Pandey e l al.42 reported that severa l bioprocesses have been developed that utili ze these raw material for the production of bulk chemicals and va lue-added fine products such as ethanol , single-cell protein (SCP) , mushrooms, enzy mes, organic acids, amino ac ids, biologica ll y acti ve secondary metabolites,

43-50 Nih 1· · f . d . 1 etc. · . ot on y t e app ,cation 0 agro-In ustna residues in bioprocess provides alternati ve substrates, but also helps solving pollution problems. Biotechno­log ica l processes, specially the SSF technique, have contributed for their utili zation . Applications of SSF to other than purely profit-driven objectives , such as environmental control, include the production of co mpost and animal feed from solid waste5

'.

Mexico and Brazil alone produce around 400, 000 metric ton of grain coffee each year and, consequently , generate great vo l umes of coffee by­products42

.5'. Due to the presence of anti­

physiolog ica l and anti-nutritional factors, coffee pulp and husk, coffee residues, are not considered an adequate substrate for bioconversion processes. Consequently, most of the pulp and husk remains unutili sed or poorly utili zed. If these tox ic consti­tuents cou ld be removed, or, at least degraded to a reasonabl y low level, it wou ld open new avenues in their utilization as substrate for bioprocesses. With this in mind, severa l authors have worked on detox ificati on of coffee pulp and husk through vari ous means. SSF has been frequent ly used for the biological detoxifi cation of coffee husk using fungal srrains52

-54

. Other app licati ons for coffee res idues were also studi ed combining both the "added value"and environmenta l control fea tures of SSF: production of aroma co mpound55

, producti on of h 565X d ·bb I· ·d59 mus roo nY - an gl ere IC aCI .

Cassava (Manihol esculenta Crantz) is widely grown in Brazil. Industrial processing of cassava tubers is mainl y done to isolate flour and starch, which generates more liquid and solid res idues (processing for flour generates solid res idues while fo r starch generates more liquid residues) . Solid residues include brown pee l, inner pee l, unusable rOOlS, crude bran, bran , bagasse and flour refu se, among which bagasse is the main residue. Cassava bagasse is made up fibrous root material and contains starch th at physicall y process cou ld not be ex tracted

(40-70%). Presently , cassava bagasse does not find suitable application, although as substrate for bio­processing. Application of cassava bagasse as substrate wou ld on one hand provide an alternati ve substrate, and on other solving pollution problem, which its di sposal otherwi se causes4

'. Such agro­industrial residues constitute promising alternati ve substrates for bioprocesses such as protein

. h 606 ' d· f 6763 ennc ment . , pro uctlon 0 aroma compounds -. , citric acid production5o.

In situ bioremedi ation processes are ga ining more and more interest since they present substanti al cost advantages and do not produce any toxic by-product as it can be the case with ex situ ph ys icochemi cal treatment24 . However, bioremedi ation is not yet uni versa ll y understood and its success is still an intensively debated issue because all so il s and groundwater are not able to sustain biolog ical growth and then, cannot be successfully bioremed iated. Therefore it is essential to carry out feasibi lity study based on pilot-test ing before starting a remed iation project in order to determine the best formulation of nutrients , oxygen transfer, pollutant bioavail ability and bacteria performance to use for the speci fic conditions encountered. In their paper, Troquet et al. 24

, compared two systems, fixed bed reactors and a rotating drum fermentor, in order to reproduce, at pilot-plant scale, columns natural biodegradation in a petroleum hydrocarbons contam inated soil laye r. The behav ior of oxygen transfer was eva luated for both systems. Best biodegradati on rates were attained with fixed bed columns in wh ich the gas fl ow passes completely through the soil layer.

Bioremediation perspectives Bioremedi ation is sti ll an immature technology ' .

Microbes are the primary stimul an t in the bioremediation of contaminated environments, how­ever current knowledge of changes in mi crob ial community is still treated as a "black box". Iwamoto and Nasu' presented a rev iew abou t bioremediation and its perspecti ves. The behavior of the target bacteria directly related to the degradation of contaminants and the changes in the microb ial communiti es during bioremedi ation, has been a chall enge for microbiologists since many environ ­mental bacteri a cannot yet be culti vated by conven­ti onal laboratory techniqucs. The application of culture-independent molecul ar biological techniques offers new opportuniti es to better understand the dynamics of mi crobi al communiti es. Fl uorescence

r

SOCCOL et al.: BIOREMEDIATION: AN ALTERNATIVE FOR SOIL & INDUSTRIAL WASTE C LEAN -U P 1043

in situ hybridization (FISH), in situ PCR, and quantitative PCR are expected to be powerful tools for bioremediation to detect an enumerate the target bacteria that are directly related to the degradation of contaminants. Nucleic acid based molecular techniques for fingerprinting the 16S ribosomal DNA (rONA) of bacterial cells, i.e. denaturating gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP), enable us to monitor the changes in bacteria community in detail. Such advanced molecular microbiolog ical techniques will provide new insights into bioremedi­ation in terms of process optimization, validation , and the impact on the ecosystem, which are indispensable data to make the technology reli able and safe .

Laboratory-scale experiments are very powerful tools to understand bioremediation in more detail, allowing the researcher to observe the main process phenomena including microbial behavior, mass transfer constraints and physical-chemical variables (PH, temperature, etc.).

Conclusions Bioremediation is an emergll1g and interdi sc i­

plinary technology that invo lves knowledge of chemical and biochemical eng ineering, ecology, statistics, microbiology , chemical, biochemical and geology, considered to be still in development. Thi s technology , generated from intensi ve experi menta­tion , comes as a response to the extensive land degradation , in order to identify the most promising way to reach the lowest possible pollution leve l. The results obtained showed that bi oremediation in its many forms , composting, landfanning, bioaugmenta­tion, wastewater biotreatment, consists on good techniques to solve the environmental probl em of water and soil pollution, resulting from accidental spilling, or the industrial activity. Even being the microorganisms are the most import part of the process , it is not very well known the mechanism through which occurs the affinity of microorgani sms to certain pollutant substance. All conclusions must be taken from experimentation. But the investigation of several industrial sectors and several kinds of pollutants, confirmed the useful and potenti al application of biotreating environmental harmful compounds that are converted into non-toxic or inert substances. With the advance of molecular microbio­logical techniques , a new tool is being added to elucidate some of these issues, and improves the solutions of some hard problem. Engineered micro-

organisms, which can be built with special abilities , are providing an important technology to degrade hard pollutants. Besides the genetic engineering can characterize most pollutant-degrading microorgan­isms and define its usage. All data research point that bioremediation is an available technology to cl ean- up liquid and so il environmental , and to treat solid and liquid industrial wastes .

Acknowledgement CRS and VTS thanks CNPq, Brazil for a

scholarship under Scientific Productivity Scheme.

References I Iwamoto T & Nasu M , C urrent bioremediati on practicc and

perspective, J 8iosci 8ioeng, 92 (200 1) I. 2 Boopathy R, Factors limiting bioremed iation technolog ies.

8ioresource Technol, 74 (2000) 63 . 3 UNEP World atlas of desertification London: UNEP/Edward

Arnold 1992 . 4 EEA In D Stanners & P Bourdeau (cds) Europe's

envirollment: Th e Dorbris assessment. Luxembourg: European Environment Agency (EEA) Offi ce for Officia l Publicat ions of the European Com muniti es. 1995.

5 Colwe ll R R, Fulfilling the promise of biotechnology 8iotechnol Advances 20 (2002) 2 15 .

6 Nout M J R. Upgrading trad iti onal biotechnol ogy processes Applications of biotechnology to trad itional fermented foods (Na ti onal Academic Press Washington DC) 1992, 11 9 .

7 Thass itou P K & Arvanitoyanni s I S, Bioremediation : a novel approach to food was te management , Trends Food Sci Technol, 12 (200 I ) 185.

8 Sturman P J Stewart P S, C unningham A B. Bouwer E J & Wo lfram J H, Engi neering sca le-up of in s itu bioremedi ation processes: a rev iew , J Comalllillall l Hydrol. 19 ( 1995) 17 1.

9 McCan y P L, Engineering concepts for in situ bioremedi ation. J Hazardous Malerials, 28 (1991 ) I.

10 Mearns A J, C lean ing o iled shores: pUlling bi oremediati on to

the test, Spill Sci Technol 8ull , 4 ( 1997) 209. II Romantshuk M, Sarand I, Petanen T, Pe lto la R, Jonson­

Vihanne M, Koivula T, Yrjala K & Haahte la K, Means to

improve the effec t o f in situ bioremediation of con taminated soil: an overview o f novel approaches, En viron Pollut , 107 (2000) 179.

12 Venosa A D, Oil spill bio remedi ation on coas ta l shorelines : A c ritique Edited by S K Sikdar & R I Irvi ne, in Riorem('C/iarion : prin.ciples and practice, Vol III , Bioremedialion lechnologies (Tech no mi c Lancaster PAl 1998,259.

13 Atlas R M, Microbia l degradation of petroleum hydroca rbons: an environmental perspecti ve Microbial Rev 45 ( 198 1) 180.

14 Atlas R M, Microbial hydrocarbon degradation- bioremediation of o il spi li s, J Chem Tecll/'lol 8iotechnol, 52 ( 199 1) 149.

15 Zobcll C E, Action of microorganisms on hydrocarbons. Bacteriol Rev, 10 ( 1946) I. '

16 Ho ff R Z, Bioremediation: an overv iew o f its development and use for o il spill cleanup Marine POllUTioll Bull 26 (1993 ) 476.

17 Swannell R P. Lee K & McDo nagh M. Field evaluations of marine o il spill bioremediation Microbial Rev 60 (1996) 342.

1044 INDIAN J EXP BIOl, SEPTEMBER 2003

18 Dojika M A. Hugenholtz P, Haack S K & Pace N R, Microbial di versity in a hydrocarbon and chlorinated­solvent-contam inated aq uifer undergoing intrinsic bioremediation , API'I EnviorOIl Microbiol, 64 ( 1998) 3869.

19 Vogel T M, Bioaugmentation as a soil bioremed iation approach , Curr Opinion Biotec/1IIol , 7 ( 1996) 3 11 .

20 Dibble J T & Bartha R. Effect of env ironmental parameters on the bi odegradation of oi l sludge Appl Enviroll Microbiol, 37 (1979) 729.

2 1 litchfield CD, III sitll bioremedi ation : bascs and practices, in M A l evin & M A Gealt eds, Biotechllology of industrial and hazardous waste (McGraw Hill USA) 1993, 167.

22 Baker K H, Bioremediation of su rface and subsurface soil s In: K H Baker & D S Hcrson eds. Biore fll ediatioll (McGraw­Hill ) 1994.

23 Boopathy R & Manning J. Surfactant-enhanced bioremedi ­ation of soil contaminated with 246-trinit roto luene in soi l slurry reactors Water Ell vironmen Res 71 ( 1999) I 19.

24 Troquet J, Larroche C & Dussap C-G, Ev idcnce for the occurrence of an oxyge n lim itati on during soi l bioremediation by solid-state fermentation , Biochem Ellg J, 3638 (2002) I.

25 Eri ckson l E. Davis l C & Narayanan M, Bioenergetics and bioremediation of contam inated soil , Th ermochimica Acta. 250 (1995) 353 .

26 Rittmann B E & Whiteman R, Biougmcntation: A coming of age Water Quality Intents, I (1994) 12, Woiciechowski 1995.

27 Barnes D, Foster C F & Hrudey S E, Food and allied industries, in Surveys il/ II/dust rial Wastewater Treatlllel/l (Pitman Advanced Publishing Program Toronto) 1984 , 376.

28 Head I M & Swannell R P J, Bioremediation of petroleum hydrocarbon contaminants in marine habitats. Curr Opinion Biotechnol,10(1999)234.

29 Balba M T. AI-Awadhi N & AI -Daher R, Bioremediation of oi l-contaminated soi l: microbiological methods for feasibility assessment and field evaluation, J Microbiol Meth , 32 ( 1999) 155.

30 Singer M E & Finnerty W R, Mi crobial metabolism of straight chai n and branched alkanes, in Altas R M Petroleum Microbiology( Maximilian Inc) 1984, I.

31 Martins J M & Mermoud A, Sorption and degradation of four nitroarometics herbicides in mono and multi -solute saturated and unsaturated soi I batch system J Contamillanl Hydrol33 (1998) 187.

32 Knack muss H-J, Bas ic knowledge and perspectives of bioelimination of xenobioti c compou nds, J Biotechnol, 51 ( 1996) 287.

33 Poll ard S J T, Hrudey S E & Fedorak PM , Bioremediation of petro leum and creosote contaminated soils: A review of constrai nts. Waste Management Res , 12 (1994) 173.

34 Baldrian P, Interation of heavy metals with wh ite- rot fungi. En zvme Microbial Technol. 32 (2003) 78.

35 Hooda P S & Alloway B, Transfer of Cd Cu Ni Pb and Zn from sewage sludge-trea ted soil into the edible parts of wheat carrots and spinach, in 3'd Internat ional Conference Con taminated Soi ls: Biotechnology of trace elemen ts INRA 087PDF Prost R, Editor 15-19, 1995.

36 lebeau T, Bagot D, JCzcquel K & Fabre B. Cadmium biosorption by free and immobili zed microorgani sms cu lt ivated in a liquid soil extract medium: Effects of Cd pH and techniques of cultu re, Sci TOlai Ellviron. 29 1 (2002) 73.

37 Bailey S E, Olin T J, Bri cka R M & Adrian D D, A rev iew of' potentially low-cost sorbents for heavy metals. Wat er Resource. 33 ( 1999) 2469.

38 Chl opecka A & Adriano DC, Influencc of zeolitc apatite and Fe-oxide an Cd and Pb uptake by crops, Sci Total EI/ virol/ . 207 (1997) 195 .

39 Vassil ev N, Toro M, Vassileva M, Azcon R & Barea J M. Rock phosphate solubili zation by immobili zed cclls of Elllerobacter sp In fermentat ion and soil conditions. Bioresollrce Tecl1l1ol, 6 1 ( 1997) 28.

40 Soccol C R & Vandenberghe L P S. Overview of applied solid-state fermentation in Brazi l. Biochem Ellg J, 13 (2003) 205 .

41 Pandey A, Soccol C R, Nigam P, Brand D, Mohan R & Roussos S, Biotechnological potential of coffee pulp and coffee husk for bioprocesses, Biochem Eng J, 6 (2000) 153.

42 Pandey A, Nigam P & Vogel M. Simul taneous saccharifi­cation and protein enrichment fe rmentation of sugar beet pulp. Biolechnol Lell, 10 (1998) 67.

43 Pandey A, Recent developments in solid state fermentation , Process Biochem , 27 (1992) 109.

44 Pandey A (Ed), Solid-siale ferlllentation (W iley Eastern New Delhi) 19943 .

45 Pandey A & Soccol C R, Bioconversion of biomass: a case study qf lignoce llulosic bioconversions in solid state fermentation , Brazilial/ Arch Biol Tecl1l1ol, 4 1 (1998) 379.

46 Pandey A, Azmi W, Singh J & Banerjee U C, 10 Biolechnology: Food f ermentalioll ed ited by YK Joshi & A Pandey Vol I (Educational Publishers & Distributors New Delhi) 1999a, 383.

47 Pandey A, Benjamin S, Soccol C R, Nigam P, Kri eger & Soccol V T, Biolechnoi Appl Biochem. 29 ( 1999b) 11 9.

48 Pandey A. Selvakumar P. Soccol C R, Soccol V T, Krieger N & Fontana J D, Appl Biochem Biolechnol, 8 1 ( 1999c) I.

49 Vandenberghe l P S, Soccol C R, Pandey A & lebeault J M. Solid state fermentation for the synthesis of citri c acid by Aspergillus niger, Bioresource Technol, 74 ( 1999) 175 .

50 va n de Lagemaat J & Py le D L, Solid-s tate fermentat ion and bioremediation: development of a continuous process fo r th_ production of fungal tann ase, Chem Eng J, 84 (200 I) 115.

5 I Brand D, Pandey A, Roussos S & Soccol C R. Biological detoxification of coffee husk by filamentous fu ngi using a so lid state fermentation system En zyme Microbial Tecl1llo1 26 (2000a) 127.

52 Brand D. Pandey A, Roussos S & Soccol C R, Mi crobial degradation of caffe ine and tannins from coffee husk. in Coffee biotechnology and qllalily ed ited by Dordrecht (Kluwer Academic Publi shers) 2000b, 393.

53 Brand D, Pandey A, leon J R. Brand I & Soccol C R. Relation between coffee husk caffeine degradat ion and respiration of Aspergillus niger in solid··state fermen tat ion, Appl Biochem Biotechnol. 102- 103 (2002) 169.

54 Soares M, Chri sten P, Pandey A & Socco l C R. Fruity fl avour production by Ceratocystis fill10rima grown on coffee husk in soli d state fermentation , Process Biochelll , 35(8) (2000) 857.

55 leifa F, Pandey A & Soccol C R, Production of mushrooms on Brazilian coffee industry residues, in Coffee biotechnology and quality Dordrecht (Kluwer Academic Publishers) 2000a. 427.

56 Leifa F, Pandey A & Soccol C R, Use of various coffee industry residues for the production of Pleurotus ostrelllus in solid state fermentation, Acta Biolechnol. 20 ( I) (2000b) 41.

SOCCOL et al.: BIOREMEDIATION: AN ALTERNATIVE FOR SOIL & INDUSTRIAL WASTE CLEAN-UP 1045

57 Leifa F, Pandey A & Soccol C R, Flaml1lulina velwipes on

coffee husk and coffee spent-ground, Brazilian Arch Bioi

Technol, 44 (200 I) 205 .

58 Machado C M M, Oliveira B H, Pandey A & Soccol C R,

Coffee husk as substrate for the production of gibberelic ac id

by fermentation, in Coffee biotechnology alld quality

Dordrecht (Kluwer Academic Publi sher) 2000, 40 I.

59 Saccol C R, !loki I, Malin B, Roussos S & Raimbault M,

Comparati ve Production of amylase and protein enrichment of

raw and cooked cassava by Rhizopus strains in submerged and

solid state fermentation, J Food Sci Technol, 3 ( 1994a) 320.

60 Saccol C R, Marin B, Raimbault M & Lebeault J M, Breeding

and growth of Rhizopus in raw cassava by solid state

fermentation , Appl Microbiol Biotechllol, 41 (1994 b) 330.

Bramorski A, Soccol C R , Christen P & Revah S, Fruit

aroma production by Ceratocystis fimbriata in static cultures

from solid agro-industrial wastes , Revista MicrobiLogia , 28

( 1998) 208.

61 Medeiros A, Pandey A, Soccol C R, Freitas R.J S & Christen P, Optimi zation o f production of aroma compounds by Kluyveromyces l1larxianus in sol id state fermentati on, Biochem £ng J, 6 ( I) (2000) 33 .

62 Favero N, Costa P & Massimino M L, In vitro up take of cadmium by basid iomycete PLeurotus ostrealus , Biotechnol Lett, 10 (1991) 701.

63 Sanglimsuwan S, Yoshida N, Morinaga T & Murooka Y, Resistance to and uptake of heavy metals in mushrooms. J Ferment Bioeng, 75 ( 1993) 112 .

64 Gabriel J, Kofronova 0, Rychlovsky P, Krenzelok M, Accumulation and effect of cadmium in the wood rotting basidiomycete, Daedalea quercina Bull Environ Coritam Toxicol, 57 (1996) 383.

65 Gabriel J, Mokrej s M, Bily J & Rychlovsky p, Accumulation of heavy metal by some wood-rotting fungi , Folia Micrubial, 39 ( 1994) 115.

66 Purkayastha R P & Mitra A K, Metal uptake by mycelia during submerged growth and by sporacarps of an edible fungus, Valvariella volvoceo, Indian J £xp BioL, 30 (1992) 11 84.