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,

:/BIOCATALYSIS-2000: FUNDAMENTALS k APPLICATIONS

15

ENVIRONMENTAL OF BIOCATALYTICAL

BIOTECHNOLOGY: AND ENGINEERING S. V. Kalyuzhnyi

THE TANDEM DEVELOPMENTS

The paper gives several examples of integrated approaches based on the tandem of biocatalytical and engineering developments in environmental biotechnology for treatment of 3 main compartments of the environment-soil, water and gas phase. The first topic
i

analyses the current situation with oil pollution of soils and water surfaces in Russia
and presents the results of field bioremediation trials on the basis of recently developed biopreparation "Rhoder". The second topic discusses the recent findings aiming to extend an applicability of anaerobic wastewater treatm-ent at temperatures as low as 4-10 DC. A performance of novel anaerobic-aerobic hybrid reactor is analysed in the third topic with regard to treatment of recalcitrant azo dye wastewater. The latter 2 topics lye within a conventional function of environmental biotechnology (so-called "end of pipe treatment") while the fourth topic dealing with the development of biocatalytical technol-

r

!..
I

ogy of H 2 S removal and sulphur recovery from polluted gaseshighlights a transformation
of this discipline into a new phase substantially sustainable production in the modern society. contributing to resource conservation and

Ii ,.
',I

Introduction The d1sarmmgly slmple defimt1on or1gmated from Alber.~ Einste~n ("Th~ environment ~s anything, which isn:t lllt: ) explams succmctly why soc1ety has so many enV1ronmental . mum. commons . . . the begmmng of the 3rd m1l1en.. Indeed, the enV1ronment 1S the "tragedy of the. " 't b 1 bd d bd Th . -1 e ongs to no 0 y an to every 0 y. IS problems at

.

..

..

..

losophy inside of environmelltal biotechnology should be holistic and this requires both a detailed knowledge about biocatalytic mechanisms involved and well designed engineering systems.
proac

Th e h

paper

es

b

ase

d

glves on

.

th t
e ., m

severa an

1
d
em

examp 0

fb

.

1

es

10ca

t 1t

0

f.

m t egra

neenng

results m the fact that ample examples of ecologIcalproblem8 or even . dIsasters
, OU8 due , efficlency. to thelr The relatlve whole

.

,rea
of

t

t men t 0f 3 mam com par t men t s 0f the enVlronmen t . .
wa t er an d gas p h ase. Th e fi rs t c h ap t er ana 1yses

developments

env1ronmental

a y 1ca an . b1otechnology

.

1

t ed

d

apeng1-

.

for

-

.

' SOl, I

ronmental

problems

lS mamly
,

"

are

encountered,

Treatment

based on blocatalytlcal
and lS reasonably

"

enVl-

meth. hlgh . enVlron-

r

mental b1otechnology, wh1ch IS currelltly ..1 .1 1. . 1 .. f b.
muustna
quantlty

,

cheapness ,. subject area

.

.

defined

h

tht~ bIggest area of
d
.

,

as

11

.

f 0

app

lcatlon

0

10cata

YS1S Wlt

regar
respect

to

overa
to the

t .t t . .th .1 11 t . f. Sl ua lon Wl 01 po u lon 0 SOlS an d wa t er 1 R "r d th It f f 11 1 b. d. laces m ussla an e resu sou -sca e loreme la . . t na' 1 on th e ba.~lS 0 f recen,. . 1 deve 1 d bloprepara s y ope " Rl d " Th d1 t t th t fi d e secon .. 10 er, ex t en d an appclap b11.presen s e b. WM t ewa t 1 . erl tyo f anacro recen n ' almmg 0 lca 1C t t t tt t 1 4 10°C A
curren
'
rea men a empera ures as ow as . per .. . ..

th e

surt'

t' lon . mgs t er f
or-

lon

t

processed

1 )(: th

ernJ

. v1ronmenta

t on "b." a t 1eas t equa 11y a.~ on tl le " t ec h no 1ogy " 10 h ' th h. t ' 1 t' tl tt tf oug m e lS onca perspec lve, Ie rea men 0 enpu

M. enVlronmen

t 1 b' t 1
a

matter.

However,

10 ec illO

1

ogy

"

wlth

th

e emp

h

. asls

Id s ou,..

h

mance
In the

of novel
thlrd

anaeroblc-aeroblc
wlth ,., regard

hybrid
to

reactor

lS dlscussed .

chapter

treatment

of recalcltrant

1

neerillg. As a consequence, the "bio" component has until recently largely been ignored and dealt with stocha.~ticalIy rather than Illechanistically, However, at present, we are facing a nUlnber of formidable environmental problems 8uch as greenhouse effect, acid rain, depletion of ozonelayer, enrichment of ground and surface waters with nutrients and recalcitrant xenobiotics, dispo8al of mlmicipalsolid and animal wastesetc. These problems can no longer be solved by a limited nulTlber of straightforward techniques, which are often a perfect illustration of Murphy's Law, i, e., they transform one problem into another often more intractable problem. Examples: one cleans water by stripping the polluti\nts into the air or removes organics from water which ~re then dumped in the soil. Hence, a particular type of waste can not anymore be treated without considering all the consequellces the environment. For instance, actifor vated sludge treatment now not only refers to the W'clter component, but also to the biosolids produced and volatile organic compounds and odour generated. Thus, the phi-

pro

bl

ems

was

.,. monopohsed

by

samtary

engl-

azo dye wastewater, Fmally. blocatalytlcal technology of H 2 S remova 1 an d su 1 hur recovery f rom po 11 d gases lS ' p ute
h.

19 Jg te

hI"

h

d

,

Spilled petroleum remediation in open water aquatories, wetlands, and soils: using novel biopreparation "Rhoder" Dlle to massive movemf~nts of petroleum from the oil-producing countries to the major oil consumersand continuous oil spills and leaks ill pipelines and storage tanks followed by.,pmoffs, approximately 35 million tons of oil enters the sea per annum [1]. Since 1 ton of oil contaminates 12 km 2 of water surface, it results in the fact thi\t 30% of the World Ocean surfi\ce are already covered hy oil film [2]. Meantime, 1 I of oil eliminates xygen from 40 m 3 of water and kills 100 million of fish larvae. Even low concentrations of oil such as 0.1 mg/l exert the death of juvenile forms of marine animals after several days of exposition and substantially inhibit the growth of microalgae [3], The toxic effectsof hydrocarbons to all forms of life

Department of Chemical Enzymology, Chemistry Faculty, Mo..cow State Univer..ity. 119899 Moscow. Russia. . V..t.ok "himi)!"
~~~ ---;~I

--


--

16
Vl:J"J.!"!n.!vJ.V"n.v.,,~~,"",'-7,"",V."'~.'~

,

,

-certificated on territory issued
delivery wet a titre

was
thus the the into

recognised
the allowing facts most account

long
lipid cell

ago
portion

and
of to oil

is usually
the escape should for be the and

ascribed On the

to the
membrane,

oil of
of

I

ration delivery No.
ly Ministry

"Rhoder" and
of

was

in

1999

for

production, (Certificate
usualof both

dissolving

cytoplasmic
[4].

I

application
Health). The

of Russia 17.08.99
form of

contents above.

basis
as one taking scale

77.99.11.515.P.4865.8.99
includes (1: a concentrated 1 w/w) with

by the Russian
"Rhoder" of cells

presented dangerous both

considered environment enormous

pollutants its high

suspension of

toxicity

of

bacteria

hydrocarbon-degrading

invasion
Russia Iraq) with total

into

biosphere.
the to oil 3rd place reserves accounted (after (62.7 for Saudi billion 295 million Arabia tons) tons and and in regard

bacteria
pared water ulators. on

of 109-1010
site by by followed

cells/mi.
of of addition

The
some

working
nutrients

solution
suspension and

is prewith biostim-

occupies

dilution

concentrated

:

:

the

1999 [5]. Due to systematic accident spills, an annual re-

oil

extraction

Sites and remediation

methods used. The follow-

leaseof oil into the environmentin Russiaaccountsfor 25 millions tonnesaccording the estimations(may be a to little bit exaggerated) "Greenpeace" Amonga variof [2].

ing oil polluted sites were used for field testing of biopreparation "Rhoder" in 1995-1999:bay of river Chernaya(Lukhovitsy, Moscow region),lakesandwetland(Vyn-

~ ~

-

gayakha, Western Siberia), lake and wetland (Ural, Western Siberia), marshy peat soil (Nizhnevartovsk, Western Siberia). Some characteristics of these sites are listen in Table 1. When necessary and possible, preliminary mechanical collection (PMC) of spilled oil on the site was undertaken before application of bioremediation technology. The latter include a spraying of the working solution on the polluted areas using pump equipment. Usually the treatment with biopreparation was repeated twice or triple with a time interval of 2 weeks. The impact of activity of indigenous hydrocarbon-degrading bacteria (HDB) wasassessed by a spraying of the working solution lacking "Rhoder" on the control areas having a similar oil pollution level. The generalisedresults of field tests are presented in Table 1. Bioremediation of open water aquatories. From some. The microbiological methodsusing both external Table 1, it is seenthat Rhoder has demonstrated very a introductions of oil-degraders cultivated ex situ and stim- high efficiency for treatment of aquatories, especiallyat low ulation of indigenous microorganislIlS (if they are present initial oil level (IOL) as in the caseof bay of river Chernaya. in necessaryconcentrations) are usually quite efficient for It should be noted that the residual oil level (ROL) after treatment of low polluted water surfaces and soils. How- 4 weeks of bioremediation of this site was only 0.04 mg/l, ever. their effects are frequently not so pronounced at a i. e., lower than the Russian PLOP (0.05 mg/l). The conhigh level of oil pollution. Besides, the low average an- centrations..pf HDB and heterotrophic bacteria firstly innual temperatures on the overwhelming majority of terricreased by 1-2 orders of magnitude at day 14 and then tory, especially where the principal oil fields are located, returned back to the initial level after an exhaustion of is another critical bottleneck for application of these meth- organic substrates in the river water. Thus, an addition ods in Russia becausebacterial oil-degrading activity drops of the external bacteria seemednot to result in substantial dramatically under temperatures below 10°C. Despite the changesof microbial community existing in the river water. above-mentionedlimitations, microbiological methods are Analogously, the initial dosageof nutrients was chosenin drawing more and more attention in our country, especially such a way that the residual level of phosphate and nitrate as post-treatment or polishing steps, due to their economic after treatment was low enough to prevent a possible euattractiveness and ability to fulfil with the stringent legisla- trophication of this bay. The both lakes in Vyngayakha tion requirementsconcerninga permissible level of oil pollu- had a high IOL (Table 1) and the thick (till 1-2 cm) oil tion (PLOP). In this chapter,the experience accumulated film was clearly seen on their surfaces. In spite of rather in 1994-1999 with application of recently developed bio- tough conditions, the triple treatment with Rhoder accompreparation "Rhoder" for spilled petroleum bioremediation panied by unusual warm weather in that period resulted in in open water aquatories, wetlands and soils is summarised. an almost complete elimination of oil pollution-the ROLs Biopreparation. The biopreparation known under were 5 and 190 mg/l in lakf~s1 and 2, respectively (Tacommercial name "Rhoder" and recently developed in ble 1). Moreover, after bioremediation both these lakes All-Russian Research Institute of oii and Gas together were certificated by the local ecological authorities as "the with Moscow State University [7] consists of two bacteria objects almost free of oil pollution". During the treatment
Rhodococcus ruber and Rhodococcus erythropolis revealof the lake;.}n Ural (Table 1), a majority of oil pollution

ety of approaches proposedfor elimination of thesespills [1], three main methods (mechanical, physico-chemicaland microbiological onesbeing applied both separately and in various combinations) are currently considered as the most perspective methods for Russian conditions [6]. Each of these methods has its advantages and drawbacks. Under fresh and abundant spills, the mechanical methods of oil collection are usually applied as a principal treatment. However, oil pollution is not eliminated completely. The physico-chemicalmethods using special reagents (detergcnts, emulsifiers, solidifiers, adsorbents, etc.) can efficiently concentrate oil pollution, but frequently they themselvesare not fairly irreproachable from the ecologicalpoint of view, e. g., collection of oil-saturated adsorbents as well as their subsequentutilisation becomessometimestrouble-

ing a synergistic action on hydrocarbon degradation under a joint application. The individual strains were isolated from oil-water mixture originating from Bondyuzhskoye oil field (Tatarstan, Russia), and the corresponding pure cultures were then deposited to the All-Russian Collection of Microorganisms (ARCM indexes are 1513-D and 1514-D, respectively) and patented [8-9]. The bioprepa-

was removed by mechanical collection (90%), i. e., the oil contamination level decreasedfrom 11 to 1.01g/l after this step. The subsequent treatment by Rhoder (twice) led to the residual oil contamination of 0.43 g/l resulting in an overall treatment efficiency of 96% (Table 1). A relatively high level of residual contamination could be mainly related to the presenceof oil polluted sedimentsaccumulatedin

-


..

BIOCATALYSIS-2000: FUNDAMENTALS

& APPLICATIONS
Table 1

"'.

~'

,~ :'1,~ Site River Chernaya

Bioremediation Area m2 100 5.000 5.000 10.000 1.900 2.000 1.000

results of "Rhoder"

field tests [6] (Pre )-treatment "Rhoder" (twice) "Rhoder" (triple) "Rhoder" (triple) "Rhoder" (triple) PMC. + "Rhoder" (twice) PMC. + "Rhoder" (triple) Treatment ffi . e. e clenCY.7. >99.9 >99.9 99 65 96 94 14-24

.

. m

Initial oil pollution the upper layer (10cm). gll 0.44 15.1 19.1 24.3

I
!

Vyngayakha: lake 1 lake 2 wetland
Ural:

.

lake wetland Nizhnevartovsk: marshy peat soil

11.0 10.5 758-828 (g/kg)

ploughing + "Rhoder" (triple)

. PMC is the preliminary mechanical collection of free oil.

this lake. These sediments served as a continuous source of oil emission to the lake water. Bioremediation of wetlands. Relatively inferior resuIts of remediation of the wetland in Vyngayakha(Table 1) can be attributed to the fact that due to specific local geological conditions it was hardly possible to apply a PMC
of free oil on this site. However, taking into account a

to apply pre-treatment, the possible strategy can includf multiple microbiological treatment with ploughing, pH ad. justing and supplementing by nutrients throughout severa years. On the aged spills (> 5 years), the oil-degradin~ activity of indigenous microflora is usually high enough t( omit an addition of biopreparations produced ex-situ. ThE
economically reasc:>nable strategy can include a stimulatioI

high 101 (> 24 gjl) and age of spill (4 years old), the resuIts look quite satisfactorily

of indigenous HDB already adapted to the site environment
Anaero

f

0

tion of PMC of spilled oil (75% removal) followed by triple treatment with Rhoder has resulted in much higher overall treatment efficiency (94%) in the caseof remediation of the wetland in Ural (Table 1). Bioremediation of soils. The inferior results of -Rhoder" bioremediation field tests obtained on the marshy peat soils in Nizhnevartovsk (Table 1) were not surprising ti\king into account an extremely high 101 (> 750 gjkg of dry matter) and age of spill (6 years). Since an over-:vhelming majorit~ of the spilled oil was adsorbed by pea.t, It was not economically reasonable to apply a PMC of 011. The pre-treatment used included only a ploughing of upper layer of contaminated area accompaniedby addition of lime (to increasepH) and nitrogen and phosphorousfertilisers. An average (for 3 lots) reduction of oil pollution was 19% (Table 1) under application of "Rhoder", while without "Rhoder" addition it was 13% (data not shown). The latter fact manifested about a high activity of indigenous HDB already developed on the contaminated site during 6 yearsand substantially stimulated by pH adjusting and nutrient addition. This supposition was further confirmed by direct counts of MPN of HDB from the lot without Rhoder addition, which were 103 and almost 106 cellsjml in the beginning and in the end of experiments, respectively (data not shown). Summarising the results presented in this chapter, one can say the following. Field tests showed a very high efficiency of biopreparation "Rhoder" for remediation of aquatories moderately contaminated by oil « 20 gjl). However, for treatment of heavy polluted aquatories (thickness of oil film> 3 mm) as well as oil spills on wetlands and grounds, the best strategy should include a preliminary mechanical collection of free oil, or application of adsorbents, or other pre-treatment methods followed by microbiological polishing step. If for some reasons it is impossible
.. V.,'nik Kh;m;ya

01 con

.1

t

amma

.

t

.

Ion

- 0 th (T bl 1)approximately t 65% removal 1 .
a e

.

n

e con rary,

app Ica-

b . wastewater IC

temperatures (4-10 C) Anaerobic treatment has several well known advan tages in comparison with aerobic treatment, especially fO treatment of high-strength wastewater - no energy need for aeration (on the contrary, generation of energy in thE form of biogas), substantially reduced nutrient require ments, high organic loading rates (01R) etc. [10]. How ever, an implementation of (;onventional anaerobic treat. ment (especially in the countries with a cold climate sud as Russial!s often hindered by the necessityof maintainin! an operation temperature-mesophilic (30-37°C) or ther. mophilic (55-60°C) which is significantly higher than am bient temperatures. This chapter discussesthe recent find ings [11-12] aiming to extend an applicability of anaerobi wastewater treatment at psychrophilic temperatures as lov as 4-10°C. Since low temperatures usually lead to a sharp de crease of the biocatalytical activity of methanogenic mi crobial consortium involved in anaerobic digestion, a strat egy in maintaining a reasonable efficiency of wastewate treatment should include an increase (as much as possible of concentration of biocatalysts inside the reactor orjanc a gradual adaptation of the consortium to psychrophili conditions. Both these approaches were combined in th, present study by using granular mesophilic sludge havin: rather high methanogenic activity and up-flow anaerob sludge bed (UASB) reactor promoting self-immobilisatio (and thus accumulation inside the reactor) of the cells c methanogenic consortium in the form of well-settled gran ules. Raw vinasse obtained by distillation of low qualit wines and diluted by tap water was us~d as feeding influ ent. The other details of experimental study are presente in works [11-12]. The perfornlance data of long-term treat ment of vinasse under psychrophilic conditions are gene alised in Table 2.

0

treatmen

t at co Id


...

,

18 ...
Performance

VESTNIK MOSKOVSKOGO UNIVERSITETA. KHIMIYA. 2000. Vol.41, No.o. ;,upplell,c"L .~ ,.&...& T"ble 2
data of long-term treatment of vinasse under psychrophilic (average values are given in brackets) [11-12].
Temperature,

'1
C ,

conditions

"C
3-5

! 1

Parameters

9-10

.
Run lb. 68-158
1:2.6 1:2.6

7-8

i Run Run days
Recycle ratio

Run la. 0-67
1:2.6

Single UASB reactor Run 2a Run 2b 159-185 197-236
1:11.6

Run 3 251-273
1:11.6

'""

OLR, g COD/lId HRT, days Influent COD, g/l Effluent COD, g/l COD removal,% Run days Reactor Recycleratio OLR, g COD/lId HRT, days Influent COD, g/l Effluent COD, g/l COD removal.%

0.3-5.1(2.7) 1.4-7.3(4.7) 3.2-4.6(3.7) 2.3-3.5(3.0) 0.8-5.1(1.9) 0.5-1.6(0.9) 0.85-0.87 0.9-1.3(1.1) 3.6-5.2(4.0) 1.2-9.9(4.2) 2.7-4.0(3.2) 3.0-3.6(3.2) 0.3-2.7(1.0) 0.5-3.6(1.8) 0.8-1.9(1.0) 0.9-1.5(1.2) 48-92(72) 48-92(60) 52-79(68) 48-70(60) Two UASB reactors in series

.

1.1-2.7(1.7) 1.14-1.17 1.3-3.1(2.0) 0.6-1.5(0.8) 15-72(57)

,

0-63 Rl R2 1:1 1:18 3.2-5.5(4.4) 0.8-4.0(2,5) 0.8-1.3(1.0) 0.8-1.2(1.0) 3.1-5.4(4.3) 1.6-3.9(2.5) 1.0-3.9(2.4) 0.4-2.8(1.2) 16-76(46) 24-80(58)

82-107 Rl R2 1:1 1:18 2.3-4.2(3.5) 1.2-3.0(2.2) 1.0-1.1(1.0) 1.0-1.1(1.0) 2.5-4.2(3.5) 1.4-3.1(2.3) 1.3-3.1(2.3) 0.4..1.9(1.0) 19-52(37) 29-78(61) Combinedsystem (Rl+R2)

122-147 Rl R2 1:1 1:18 2.0-2.7(2,5) 1.5-2.2(1.7) 0.8-1.0(0.9) 0.8-1.0(0.9) 1.9-2.6(2.4) 1.1-1.9(1.5) 1.1-1.9(1.5) 0.3-1.2(0.7) 25-52(37) 43-74(53) 1.0-1.4(1.3) 1.6-2.0(1.8) 60-86(71)

OLR, g COD/lId 1.6-2.8(2.2) 1.2-2.1(1.8). HRT, days 1.6-2.5(2.0) 2.0-2.2(2.0) COD removal,% 36-91(78) 42-89(76) Run la-non-preacidified influent; run lb-preacidified influellt.

.

t

One stage U ASB psychrophilic treatment. Dtlring run la (10°C), when non-preacidified influent wa.'J treated. an OLR was increl\8ed stepwise to 4-5 g COD/lid with a total chemical oxygen demand (COD) removal of around 70%. (Table 2). A significant presence of propionate (predominant component) and acetate was observed in the efflu& ents. However, only traces of sugars, ethanol and butyrate were detected in the reactor liquor, while the headspace gas hydrogen concentration was negligible. These facts clearly demonstrate that low temperatures affect the various . stages of anaerobic digestion differently, with propionate conversion becoming the rate-limiting step [13]. It should
be also noted that a substantial increase

above-mentioned supposition about the existence of mass transfer limitations inside the psychrophilic sludge bed. Decrease of temperature during run 2a to 7 °C did not result in deterioration of reactor performance though the OLRs were somewhat lower (around 4 g COD/lid) than those applied during run 1b (Table 2). In order to decrease mass transfer limitations, the recycle ration was increased during run 2b (days 197-236, Table 2). As expected, an almost 4 times increase of Vup resulted in a better VFA removal though a total COD removal efficiency slightly de~reased compared to run 2a. This was mainly due to an mcreased.~ludge washout beca~se small sludge ~g~regate~

(- 20%) of sludge

were c~ntmu?usly accumulated m the effluent ~eclplentvessel dunng this run. A further decrease of workmg temperatur~ to 4 °C was accompanied by a decrease of OLR imposed
dunng run 3 (days 251-273,

bed height had occurred over this run that was primarily due to a substantial growth of acidogens in the reactor , bec a use fl a u ff y ou t er

seen gates.

un

such types of aggregates . can provoke sludge . . , flotation and create mass transfer..hmltatlons for substrates . , .. of proplonate-degradmgand acetlcla.'Jtlc bactena which are . .

. Smce

der

. . microscopiC

t. 0 serva Ions

b

1 ayer

. covenng

th

0

f th

1d e s u ge aggre-

e granu

1 es was

all

reactor
O

performance washout

was slmllar

Table ..

2).

In

general, .

the

over.

A sludge

. .. usually located m the central . part of aggregates, It was decided to b apply preacldlficatlon of wastewater .in order h. ,
I

dunng thIS run. It was becausea . t 1 d 1 . td . aggrega es were a rea y e lmma e 2b M lcroscoplC 0 bserv,~ t . . . run. Ion

was also ob8erved

to that dunng run 2b . but It tended to deceasE

maJont y 0f fi ne s1 dgf u f th t d. rom e reac or urm~

'

'

0 f th e s1u d ge t a k en a t th e en d 0 f run 3 sh owe d an overw h e1mmg pre d ommance 0 . .

fl uff y 1 t . lrregu 1 forms and I' I arge aggrega es (4- 5 mm ) WIth . ar
looked like flocculent one. Such evolution of the sludge car. be attributed to the fact that the reactor influent was nol

to ac. i~ve e~ter COD removal. Howev~r, feedmg wI~h preacidlfied vmasse (run 1b, Table 2) dId not result m

!

a~y enhancement ?f COD removal with the effluent propionate concentratIons o:te~ ex~eeded 1.5 g COD/I, In o.rder.to have a deep~~mslght mto the processeso~currmg m the psychrophllic UASB reactor, the sludge kmetic ch~~acteristicswere assessedin situ, i. e., under reactor condItions (days 120-138). Apparent half saturation con..,tants Km for all the substrates tested were found (data not shown) to be greater than 1.0 g COD/l at the imposed up-flow liquid velocity (Vup) of 0.1 m/h, which supports the

completely acidified by preacidification procedure applied e. g., sometimes quite noticeable concentrations of ethano (till 2 g COD/I) and sugars (till 0.6 g COD/I) entered t< the reactor stimulating a development of fluffy acidogenil biomass which deteriorated a sludge quality. Thus, a con trol of preacidification efficiency seems to be essentialfo a stable p;~treatment proceS8of winery wastewater at 10' temperatures. Two stage U ASB psychrophilic treatment. In or

--.

~~

~


.

BIOCATALYSIS-2000: FUNDAMENTALS der to control preacidification

& A1'1'Ll\';AJ.lV!~" erties of azo dyes dictate the anaerobic-aerobic sequence in
designing an efficient biomineralisation process. Two separate treatment steps are usually applied for this purpose.

_I
-

of wastewater with the aim

to enhance a COD removal two UASB reactors were combined in series. Reactor R1 ~ainlY served a.~ preacidificator

-~

to generate VFA for feeding reactor R2. High recycle ratio (1: 18) was applied in reactor R2 in order to decrease mass transfer limitations while recycle ratio in reactor Rl was kept at low level (1: 1) because diffllsionallimitations are not very important for fast acidogenic step. The sludge from run 3 consisting predominantly of fluffy large aggregates (see above) was used as a seed for both the reactors. Analysing the results obtained during two-stage UASB pretreatment (Table 2), one can say the following. A combined system with two reactors significantly better operation staremoval efficiencies and in series has demonstrated higher bility compared to : single U:A.SB t~ea~ment at ~emperatures as low as 4-10 C. Any difficultIes m a combmed systern performance including sludge lifting or heavy washout have no~ been observed at all. It should be not~d, however, that a sIngle UASB reactor was operated at hIgher OL~ . .. . (but wIth ~reacldlfied wast~water) than t.h~ OL~ Imposed ?n a combm~d system treatmg non-preacldlfied wastewater If one takes litO acc~unt. the overall volume of both reactors. Thus, an apphcatlon of two UASB reactor system
. .

In order tC1~ptimise the t~eatment process and ove.rall economics of the correspondmg technology, we combIned the anaerobic and aerobic phases into one single unit called the anaerobic-aerobic hybrid reactor (AnAHR) in this study (Fig. 1 a ). The advantages of this innovative design include reduced aeration costs and lower space requirements while offering substantial mitigation of a broad spectrum ofrecalcitrant xenobiotic contaminants (not only azo dyes) found in industrial wastewaters. This chapter discusses the performance of the mesophilic (30°C} AnAHR using the azo dye Siriusgelb (Fig. Ib) and ethanol as donor of reductive equivalents (Fig. 2) [16]. The concentrations of Siriusgelb and ethanol were 0.3 and 0.82 g COD/I, respectively. It should be noted that throughout the entire experimental run, only traces (if any) of ethanol and acetate (very rarely) dt tdI ' were e ec e n the U Pp er Part of the anaerobic zone of the AnAHR. This suggests that the conversion of ethanol to methane was already complete in this zone and the measured COD content of the slLmples taken from the upper
t f th '" b . t t 11 as those of the par 0 e anaero IC com par men as we

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a single UASB reactor operating at psychrophilic temperatures seems to need at least a partial preacidification of wastewater in order to ensure its more-or-less stable operation. Concluding this chapter, it should be ~oted that anaerobic treatment in high rate reactors like UASB reactors is feasible at temperatures as low as 4-10°C. However, substantial mass-transfer limitations for the soluble substrates inside the reactor sludge bed were encountered. Therefore, an application of higher recycle rations is essential for enhancement of UASB treatment under psychrophilic conditions. The produced anaerobic effluents were shown to be efficiently post-treated aerobically-final effluent COD concentrations were around 0.1 ~/l [12]. A successful operation of the UASB reacto~s at qUIte l~w t~mpera~ures (4-10°C) op~ns good perspectlve.s for apphcatlon of l11gh~rateanaerO?IC treatme~t at ambIent temperatures, e. g. m south reglons of RussIa.

During the first 18 days, when the azo dye loadIng ratE (ADLR) was 0.09 g COD/I/day using a HRT (hydrauli< retention time) of ~pproximILte~y 3.4 days (Fi.g. 2a), az( dye treatment efficIency (TE) m the anaerobIc compart. ment was 51% and the overall TE of the AnAHR Wal 71% (Fig. 2b). After an in(:rease of ADLR to an aver. age value of 0.18 g COD/l/dILY for the period from day 1~ to day 32 (Fig. 2a), azo dye anaerobic and overall TEl dropped slightly and were on average 50 and 64%, re, spectively (Fig. 2b). In the final stage of this experi. ment (day 33 onwards), the ADLR was further increased t< 0.3 g COD/I/day keeping the HRT around 1 day (Fig. 2a) This resulted in a further drop of both TEs-44 and 56Oj (on average) for the anaerobic compartment and the en tire AnAHR, respectively (Fig. 2b). Negligible absorban cies at 37:5.nm (maximum absorbance of Siriusgelb) wer observed in the reactor effluent throughout the entire ex perimental run indicating complete decomposition of thi azo d y e.
ent dId not

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colour compared to the IntensIve brownish-yellow
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duced world-wide [14]. Approximately 10-15% of overall production is released into the environment mainly via wastewater [15]. This is very dangerous because some of the azo dyes or their breakdown products have a strong toxic, mutagenic or carcinogenic influence on the living organisms;

:,'

therefore,the corresponding wastewaters shouldbe treated before discharge. However, a majority of azo dyes are quite resistant to biodegradation under aerobic conditions

Azo dye

Efnuent

Fig. 1a. The experimental set-lIp for biomineralisation of azo dyes in the AnAHR.

and easily pass through conventional aerobic wastewater treatment systems. On the other hand, azo dyes are read-

ily decolourised splitting the azo bond(s) in anaerobic by
are more susceptible to biodegradation under aerobic conditions rather than under anaerobic conditions. These prop-

COONs

environments. turn, the anaerobic In breakdown products 011

h V

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Fig. 2. Operation performance of AnAHR treating a Siriusgelb (0.3 g COD/I) synthetic wastewater supplemented with ethanol (0.82 g COD/I).

of influent. A transient accumulation of intermediate of Siriusgelb decomposition-5-aminosalycilic acid (5-ASA)was detected in the anaerobic compartment of the AnAHR but not in the effluent. The 5-ASA concentrations peaked (till 0.06 g COD/I) immediately after increasesof ADLR and then gradually decreasedif the ADLR was kept constant. This observation suggestsa stepwise adaptation of anaerobic sludge for decomposition of 5-ASA. As can be seenin Fig. 2b, a majority of the azo dye COD was removed in the anaerobic compartment and the aerobic section had a relatively minor impact on the overall TE. The aerobic removal, as a percentage of the influent, varied between 20 and 30%. Such low TEs achieved in the aerobic step as well as effluent colouring can be attributed to the fact that the breakdown products of anaerobic Siriusgelb decomposition (5-ASA and 1,4-phenilenediamine)are readily autooxidisedto coloured polymeric products upon exposure to air [17]. These autooxidation products are often complex humic compounds that are non-biodegradable. Incomplete recovery of ammonia (data not shown) also supports the above-mentionedsupposition about the inclusion of generated aromatic amines in these persistent polymeric products. Thus, an innovative reactor construction where the anaerobic and aerobic phases were combined in one single of unit called an AnAHR is proposed for the treatment as persistent xenobi.azo dyes . well as other aerobically otIC contamInants. The performance of the AnAHR was tested with a synthetic wastewater containing Siriusgelb and ethanol as co-substrate at 30°C. Almost complete decolouration of the influent and 56% removal of the azo dye COD was achieved using a HRT of 1 day and volumetric loading rate of 0.3 g azo dye COD/I/day. The effluent contained no ethanol or acetate and its COD content could be attributed to the presenceof non-biodegradable autooxidation products of Siriusgelb breakdown intermediates. Further researchis needed to assessthe feasibility of this reactor concept for treatment of industrial wastewater containing persistent compounds.

reactions in a liquid phase, physical absorption and direct chemical conversion have several evident drawbacks: high reagent consumption, equipment corrosion, application of high temperature and pressure, etc., resulting in high process costs-250-750$/ton of removed sulphur [19]. This chapter highlights the development of substantially cheaper alternative-biocatalytical technology for treatment of H 2S polluted gases. The schematic representation of the process proposed is shown ill Fig. 3 [18]. Briefly, In the scrubber, the H2 S containing gas comesinto contact with a slightly alkaline (pH 8.0-8.5) scrubbing solution whereabsorption of..If 2S takes place. Scrubbing liquor then passe to the bioreactor containing immobilised bacteria of genus Thiobacillus where a soft oxidation of sulphide into elemental sulphur accompanied by regeneration of alkalinity proceeds. Solid sulphur is removed and the liquid is returned to the scrubber for absorption of the next portion of H2 S. Since the successof elegant technological schemepresented in Fig. 3 is determined (in major extent) byefficiency ofbioreactor, significant efforts were put on optimisation of its construction and produ(:tivity [18]. The crucial point is that the bacteria of genus Thiobacillus used in the process oxidise sulphide not only into sulphur but also into sulphate:
HSHS+ + 0.502 202
~ ~ SO

+ OH-, + H+.

(1) (2)

S024

Biocatalytical technology for H 2 S removal and sulphur recovery from polluted gases
Biogas usually contains around 1 vol.
%,

Obviously, the reaction (2) is highly undesirable for the processunder development becauseit leads to expenditure of alkalinity of the liquid phase and formation of hardly removable dissolved product (sulphate). To suppress this rea(:tion, oxygen-limiting conditions and high sulphide loading rates should be imposed on the system [18]. In an engineering context, the various reactor constructions (conventional CSTR, reactor with external aerated loop, gas-lift) were tested on the laboratory level [18]. Currently the best construction consists of an automated close (with respect to gas-phase)gas-lift reactor equipped with on-line sensorsfor measuring dissolved oxygen, sulphide and pH. The electric signals from these sensorswere transferred to a programmable data logger system. A personal computer programmed to function as a terminal emulator was used
to communicate with the data logger and to control the

of

H2

S,

while

natural gas can contain till 15 vol. % of H2 S [18]. Conventional technologiesfor treating such gasesbasedon chemical

feeding pumps. Using this highly controlled reactor and pure oxygen (instead of air), 94-98% efficiency of sulphide


BIOCATALYSIS-2000: FUNDAMENTALS Gas free of HIS

& APPLICATIONS

.

21
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H S + OH- -I H S- + H I 0 : Gas with H:S
,

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S,o..+OH.. Sulphur

:

Fig, 3. Schematic representation of biocatalytical reagent less method for H 2 S removal from polluted gases.

conversion into elemental sulphur under sulphide loading rates as high as 15 g S/l/day was achieved [18]. Finally, the manifest advantages of the proposed technology compared to the conventional methods should be underlined: practically reagent less character (some salts are necessary for bacteria); cheapness; practically closed cycle and minimum of wastewater; sole process product (sulphur) can be readily re-used (for sulphuric acid production); ambient temperature and pressure for the process making it safe. Concluding remarks The presented examples clearly demonstrate that by creating optimal growth conditions for microorganisms in proper designed engineering systems, conversion rates can be significantly increased resolving many problems of environmental biotechnology. Moreover, during last decade this discipline has matured from its conventional function (so-called "end of pipe treatment") to a new phase substantially contributing to resource conservation and sustai~able production in the modern society. The discussed above biocatalytical technology for H2 S removal and sulphur recovery from polluted gases is a typical example of this. Acknowledgements The financial supports of INTAS (Grants 96-1809 and
96 2045) N th I d R h0 . t. (G t 97 ,e er an s esearc rgamsa ion ran 29925) IPP f US D t t fE (G t , program 0 epar men 0 nergy ran

3. Murygina, V.P., Arinbasarov, M. U.j and Kalyuzhnyi, S.V. (1999) Ecology and industry of Russia, No.8, 16-19. 4. Currier, H.B. and Peoples, S.A. (1954) Hilgardia, 23, 155-174. 5. Goskomstat RF (2000) Russia in figures p.OOO:official statistics, Gosko~stat RF Press, Moscow. 6. Mlll"ygina, V., Arinbasarov M., and Kalyuzhnyi S. (2000) in Proc. of the 4tl1 Intern. Symp. on Environmental Biotechrlology (Hartmans, S. and Lens, P., eds.), Noordwijkerhout, the Netherlands, pp. 319-322. 7. Patent RF No. 2090697 (1997). 8. Patent nr No. 2069492 (1996). 9. Pat.ent RF No. 2069493 (1996). 10. Lettinga, G. (1996) Wat. Sci. Technol., 33(3), 85-98. 11. Kalyuzhnyi, S.V., Gladchel1ko,M.A., Sklyar, V.I., Kurakova, O.V., and Shcherbakov, S.S. (2000) Environ. Technol., 21,919-925. 12. Kalyuzlulyi, S.V., Gladcllenko, M.A., Sklyar, V.I., Kizimenko, Ye.S., and Shcherbakov S.S. (2000) Appl. Biochem. Biotcchnol (submitted for publicati~n). 13. Rebac, S. (1998) Psychropl1ilic anaerobic treatment of low strength wastewaters, Ph. D. thesis, Wageningen Agricultural University, The Netherlands. 14. Carliell, C.M., Barclay, S.J., Naidoo, N., Buckley, C.A., Mulholland, D.A., and Senior, E. (1995) Water SA, 21(1), 61-69. .
15. \ Tan, . Lettmga, N.C.G., G., Bor~er, and

325733A G2) R . F d t. fB. R h , USSlan esearc (G t 99 15 96064) R . oun .. ion 0fS .aslC d T h Ma ra~ -, USSlan m~stry 0 c!ence an ec -

--

IAWQ C f "W M ... t. d E d f P. on erence aste Inlmlsa Ion an n 0 Ipe Tr . Ch . I dP h . I rd ." eatment In emlca an etroc emlca In ustnes
(BuitroI1j" G. and Macarie, H., eds.), Merida,
"

A., . FIeld,

Slenders, J.A. (1999)

P.,

.. m

Svltelskaya, ProceedIngs

A., of

Mexico,

nologles (G~ant 99-419), Luk?ovltskaya oll-!>ro~~ct st?rageI enterprise (Moscow provmce), compames LUKollU f "" b """. ra ne tegas, Noya rskneftegas, Green (Nlzhnevardd tovsk) and "Orenburggasprom" are gratefully acknowle ge . References 1. Rosenberg, E. and Ron, E.Z. (1996) in Bioremediation: principles and applications (Crawford, R.L. and Crawford, D.L., eds.), Cambridge University Press, New York, USA, pp. 100-124. 2. Zhanovich, A.V., Gridin, O.M., and Gridin, A.O. (1995) in Proc. of the All-Russian Conference on Ecological Problems, Moscow, pp. 3-6.

16 Kal

.

pp. 227-234. I .S t hI yuz UlYl, . and SkIyar,. V (2000) Wa. SCI. J.ec no., 41, No. 12, 23-30.
",

17. Razo-FI ores,.E (1997). B 10 ransf orma t Ion and b10 egra a.t " d d Lion of N-.,ubstituted aromatics in methanogenic granular slt¸dge,Ph. D. Thesis, Wageningen Agricttlture University, The Netherlands. 18. Kalyuzhnyi, S.V. and Fedorovich, V. V. (2000) Ecology and industry of Russia, No.2, 33-36. 19. Lagas, J .A. (2000) in Environmental technologies to treat sulfur P9'lution: principles and engineering (Lens, P. and Hulshoff Pol, L., eds.), IWA Publishing, London, pp. 237264.

11

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